Life Extension Magazine®

Issue: Nov 2009


Scientifically reviewed by: Dr. Gary Gonzalez, MD, on January 2021.

A double-blind, randomised- placebo, controlled, parallel group, multicentre, flexible-dose escalation study to assess the efficacy and safety of sildenafil administered as required to male outpatients with erectile dysfunction in Korea.

The efficacy and safety of sildenafil was evaluated in a randomiSed, double-blind, placebo-controlled, flexible-dose study in Korean men aged 28-78 y with erectile dysfunction (ED) of broad-spectrum aetiology and more than 6 months duration. A total of 133 patients were randomised at six centres in Korea to receive either sildenafil (50 mg initially, increased if necessary to l00 mg or decreased to 25 mg depending on efficacy and tolerance) (n=66) or matching placebo (n=67) taken on an ‘as needed’ basis l h prior to anticipated sexual activity for a period of 8 weeks. At the end of this time, the primary efficacy variables relating to the achievement and maintenance of erections sufficient for sexual intercourse, and the secondary efficacy variables, which included: (1) the five separate domains of sexual functioning of the International Index of Erectile Function (IIEF) scale, (2) the percentage of successful intercourse attempts, and (3) a global assessment of erections, were all statistically significantly improved by sildenafil in comparison with placebo (P&<0.0001). Treatment-related adverse events occurred in 56.1% of patients receiving sildenafil and 20.9% receiving placebo. The most common adverse events with sildenafil were vasodilatation (flushing), headache and abnormalities in colour vision (31.8, 22.7 and 6.1% of patients, respectively), and most were mild in nature. The efficacy and safety of sildenafil in this population of Korean men appears similar to that reported in other studies in western populations.

Int J Impot Res. 2003 Apr;15(2):80-6

Viagra—the first oral treatment for impotence that is not lacking in fatal effects.

Impotence, a common problem especially among older men, can now be treated with Viagra, This oral pill, unlike previous approved treatments mostly involving local injections, does not directly cause penile erection, but increases response to sexual stimulation. It acts by enhancing the relaxant effects of nitric acid on smooth muscle, and thus increases blood flow to certain areas of the penis, leading to erection. It has been evaluated in many randomized trials and in all was more successful in inducing erection than placebos. The most common side-effects include headache, flushing and indigestion, but there have also been reports of fatalities. We describe a 75-year-old man who had an acute myocardial infraction in the past and who had maturity-onset diabetes and hypertension. In the week prior to admission he had a cardiac scan following a few weeks of exacerbation of anginal pain for which he had been taking nitrites. He took a Viagra pill without prescription or medical advice and 2 hours later, during intercourse with his wife, developed audible respiratory distress and lost consciousness. His wife started cardiac massage but not mouth-to-mouth breathing. The emergency team found ventricular fibrillation and gave 5 electrical shocks and amines and atropine. He remained unconscious, but his pulse returned and he was hospitalized. He then had several generalized convulsions treated with i.v. valium. 20 minutes after admission there was asystole and all attempts at resuscitation failed. Cardiovascular status must be considered prior to prescribing Viagra, and the associated risk evaluated.

Harefuah. 1998 Jul;135(1-2):1-2, 88

Sildenafil citrate for erectile dysfunction in men with diabetes and cardiovascular risk factors: a retrospective analysis of pooled data from placebo-controlled trials.

OBJECTIVE: Cardiovascular (CV) risk factors are associated with an increased risk of erectile dysfunction (ED). In men with diabetes mellitus (DM), pooled from clinical trials of sildenafil treatment for ED, this retrospective analysis determined efficacy and safety, overall and in subgroups with additional CV risk (i.e., hypertension, dyslipidemia, and smoking). RESEARCH DESIGN AND METHODS: From the manufacturer’s database of worldwide research, 12-week data from men with DM were pooled from randomized, double-blind, placebo-controlled trials of flexible-dose sildenafil (25, 50, or 100 mg, PRN) for ED. MAIN OUTCOME MEASURES: Question 3 (achieving an erection), question 4 (maintaining an erection), and the Erectile Function domain of the International Index of Erectile Function; percentage of successful intercourse attempts according to patient event logs; and response to a global efficacy question (GEQ). Differences between groups were determined using logistic regression (percentage of responders according to GEQ) and analysis of covariance (all other outcomes). RESULTS: Inclusion criteria were met by 11 trials and by 974 men with DM and ED who were randomized to placebo (n = 482) and sildenafil (n = 492) within the selected trials. For all outcomes, overall and regardless of additional CV risk, the benefit was greater for sildenafil versus placebo (p < or = 0.0001), including 3-fold more men responding that sildenafil treatment improved their erections (62% vs. 18%) and a more than doubling of the mean +/- standard error percentage of successful sexual intercourse attempts (52.6 +/- 5.0 vs. 22.4 +/- 5.1). Adverse events were mild to moderate and included (sildenafil vs. placebo) headache (5% vs. 2%), flushing (7% vs. 2%), and dyspepsia (4% vs. 0%), which is consistent with the profile in the general population of men treated with sildenafil for ED. CONCLUSION: This retrospective analysis of pooled data showed that sildenafil was well tolerated and improved erectile function and intercourse success in men with ED and DM, regardless of additional CV risk factors.

Curr Med Res Opin. 2006 Nov;22(11):2111-20

Update on the relationship between sexual dysfunction and lower urinary tract symptoms/benign prostatic hyperplasia.

PURPOSE OF REVIEW: Lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) is a condition that commonly affects older men and is often associated with sexual dysfunction. Recent evidence of an association between LUTS/BPH and sexual dysfunction will be reviewed, as well as the effects of pharmacological treatment options for symptomatic LUTS/BPH on sexual function. RECENT FINDINGS: Large-scale epidemiological studies conducted worldwide have provided strong evidence for an association between LUTS, erectile dysfunction and ejaculatory dysfunction. In multivariate analyses controlling for age, comorbidities, and lifestyle factors, LUTS have been clearly demonstrated to be an independent risk factor for erectile and ejaculatory dysfunction. Various pathophysiological mechanisms have been proposed for the association between LUTS and male sexual dysfunction. These include autonomic hyperactivity, alterations in Rho/Rho kinase pathway, endothelial (nitric oxide synthase/nitric oxide) dysfunction, pelvic ischemia, and age-related hormone imbalances. Owing to the link between LUTS/BPH and male sexual dysfunction, patients presenting with one of these conditions should be routinely screened for the other condition. In addition, because medical and surgical treatments for LUTS/BPH are commonly associated with sexual side effects, patients with LUTS/BPH should be monitored for treatment-related sexual outcomes. SUMMARY: LUTS/BPH is an independent risk factor for sexual dysfunction in aging men. Further studies are needed to define the mechanism(s) underlying the link between LUTS/BPH and male sexual dysfunction. Additional studies of combination therapy for LUTS/BPH, sexual dysfunction, and other age-associated comorbidities are needed to establish new approaches to the optimal management of these conditions in aging men.

Curr Opin Urol. 2006 Jan;16(1):11-9

Effects of long-term oral administration of L-arginine on the rat erectile response.

PURPOSE: Nitric oxide (NO), the neurotransmitter responsible for mediating penile erection in the rat, is synthesized from L arginine by nitric oxide synthase (NOS) in a reaction blocked by L-NAME (N-omega-nitro-L-arginine methyl ester). To determine whether dietary supplementation of L-arginine can stimulate penile erection and whether ancillary pathways for penile erection may exist, a series of experiments were conducted in the Fischer 344 rat. MATERIALS AND METHODS: Adult male (5 month old) and aged (20 month old) rats were fed L-arginine (2.25%) and L-NAME (0.7%) dissolved in tap water for 8 weeks. Animals (n = 6) underwent electrical field stimulation (EFS) of the cavernosal nerve to induce erection and both maximal intracavernosal pressure (MIP) and mean arterial pressure (MAP, mm. Hg +/- SEM) were measured. Tissue and serum levels of L-arginine were measured by an automated amino acid analyzer. Penile eNOS (endothelial) and nNOS (neuronal) content were measured by western blot densitometry. Total penile NOS enzyme activity was measured by the L-arginine to L-citrulline conversion assay. RESULTS: The L-arginine fed animals demonstrated a significant increase in EFS-induced MIP when compared to the controls in both the adult (104 +/- 4 vs. 86 +/- 6, p = 0.04) and aged (87 +/- 5 vs. 66 +/- 4, p = 0.02) animals, without changes in MAP. L-NAME virtually abolished the MIP in adult rats (8 +/- 3, p < 0.0001), while increasing the MAP (186 +/- 8, p < 0.0001). Serum and penile tissue levels of L-arginine were increased by 64-148% in all groups compared to control animals. Penile eNOS and nNOS content remained unchanged in control and treated animals. Penile NOS activity was increased nearly 100% in the L-arginine treated groups vs. controls. CONCLUSIONS: Long-term oral administration of supra-physiologic doses of L-arginine improves the erectile response in the aging rat. We postulate that L-arginine in the penis may be a substrate-limiting factor for NOS activity and that L-arginine may up-regulate penile NOS activity but not its expression. The blockade of penile erection by EFS with L NAME suggests that if ancillary corporeal vasodilator mechanisms develop, a basal level of NO synthesis is still required for activation and relaxation of the corporeal smooth muscle. These data support the possible use of dietary supplements for treatment of erectile dysfunction.

J Urol. 1997 Sep;158(3 Pt 1):942-7

Treatment of erectile dysfunction with pycnogenol and L-arginine.

Penile erection requires the relaxation of the cavernous smooth muscle, which is triggered by nitric oxide (NO). We investigated the possibility of overcoming erectile dysfunction (ED) by increasing the amounts of endogenous NO. For this purpose, we orally administered Pycnogenol, because it is known to increase production of NO by nitric oxide syntase together with L-arginine as substrate for this enzyme. The study included 40 men, aged 25-45 years, without confirmed organic erectile dysfunction. Throughout the 3-month trial period, patients received 3 ampoules Sargenor a day, a drinkable solution of the dipeptide arginyl aspartate (equivalent to 1.7 g L-arginine per day). During the second month, patients were additionally supplemented with 40 mg Pycnogenol two times per day; during the third month, the daily dosage was increased to three 40-mg Pycnogenol tablets. We obtained a sexual function questionnaire and a sexual activity diary from each patient. After 1 month of treatment with L-arginine, a statistically nonsignificant number of 2 patients (5%) experienced a normal erection. Treatment with a combination of L-arginine and Pycnogenol for the following month increased the number of men with restored sexual ability to 80%. Finally, after the third month of treatment, 92.5% of the men experienced a normal erection. We conclude that oral administration of L-arginine in combination with Pycnogenol causes a significant improvement in sexual function in men with ED without any side effects.

J Sex Marital Ther. 2003 May-Jun;29(3):207-13

Sperm parameters in male idiopathic infertility after treatment with prelox.

The diagnosis of male infertility is determinate after assessment of sperm quality and clinical study. In nearly 30% of the cases nevertheless detailed clinical and laboratory study it can’t be discovered the cause and on the bases of exclusion criteria set the diagnosis idiopathic infertility. The object of our study was investigation of the group patients (n=50) with idiopathic infertility treated with Prelox and to be studied its effects on spermatozoa parameters. MATERIAL AND METHODS: The study design was double-blind, placebo-controlled, cross-over, randomized study, including introduction period (1 month), two therapeutic periods (each one of 1 month) separated with 1 month wash out period and concluding period of 1 month. There was applied a new method for treatment with mechanism of action stimulation the production cGMP of spermatozoa endothelial nitric oxide synthase (eNOS). This is not surprising achieving results show improvement of sperm quality. The methods of the study were: 1. Assessment of the conventional semen analysis (according the criteria of WHO, 1999). 2. Spermatozoa function tests. 3. Spermatozoa-cervical mucus penetration tests. RESULTS: The obtained results showed improvement of sperm quality, in the middle-aged men the therapeutic answers was better than in younger. In conclusion the therapy with Prelox improve sperm parameters in men with idiopathic infertility. Pycnogenol (one of the constituents of Prelox) has powerful antioxidative influence ameliorating spermatozoa function.

Akush Ginekol (Sofiia). 2007;46(5):7-12

Improvement of erectile function with Prelox: a randomized, double-blind, placebo-controlled, crossover trial.

In a randomly allocated, double-blind, placebo-controlled, crossover design, 50 patients with mild to moderate erectile dysfunction (ED) were treated for 1 month with placebo or a combination of L-arginine aspartate and Pycnogenol (Prelox). Patients reported sexual function from diaries. Testosterone levels and endothelial NO synthase (e-NOS) were monitored along with routine clinical chemistry. Intake of Pycnogenol for 1 month restored erectile function to normal. Intercourse frequency doubled. e-NOS in spermatozoa and testosterone levels in blood increased significantly. Cholesterol levels and blood pressure were lowered. No unwanted effects were reported. Prelox is a promising alternative to treat mild to moderate ED.

Int J Impot Res. 2008 Mar-Apr;20(2):173-80

Improvement of seminal parameters with Prelox: a randomized, double-blind, placebo-controlled, cross-over trial.

In a randomly allocated, double-blind, placebo-controlled, cross-over design, 50 infertile patients were treated for 1 month with placebo or a combination of l-arginine aspartate and Pycnogenol (Prelox). Semen samples were examined at 4 week intervals according to WHO criteria. Treatment with Prelox increased significantly the semen volume, concentration of spermatozoa, percentage of motile spermatozoa and percentage of spermatozoa with normal morphology compared with placebo. The placebo had no influence on the parameters of seminological analysis. Intake of Pycnogenol for 1 month improved the fertility index to normal values. After treatment, the fertility index decreased again to infertile status. No unwanted effects were reported. Prelox seems to be a promising alternative to treat patients with mild infertility.

Phytother Res. 2009 Mar;23(3):297-302

Nitric oxide and penile erectile function.

The discovery of nitric oxide (NO) as an intercellular messenger or neurotransmitter opened a new era for identifying the important mechanisms underlying physiological and pathophysiological events in autonomically innervated organs and tissues; it also provided the way for development of new therapeutics based on a novel concept of molecule and cell interaction. Endothelium-derived relaxing factor (EDRF) discovered by Furchgott and Zawadzki has been proved to be NO, a labile gaseous molecule, that modulates vascular tone, platelet aggregation and adhesion, and vascular smooth muscle proliferation. Later, NO was determined to act as a non-adrenergic, non-cholinergic (NANC) neurotransmitter of postganglionic parasympathetic nerve fibers, innervating a variety of smooth muscles including the penile corpus cavernosum (CC). The nerve is called “nitrergic” or “nitroxidergic”. Although CC sinusoidal endothelial cells also produce and liberate NO in response to chemical and possibly physical stimuli, roles of neurogenic NO in penile erection appear to be more attractive and convincing. NO is formed from L-arginine via catalysis by NO synthase (NOS) isoforms, neuronal (nNOS), endothelial (eNOS), and inducible NOS. NO from nerves and possibly endothelia plays a crucial role in initiating and maintaining intracavernous pressure increase, penile vasodilatation, and penile erection that are dependent on cyclic GMP synthesized with activation of soluble guanylyl cyclase by NO in smooth muscle cells. Erectile dysfunction (ED) is caused by a variety of pathogenic factors, particularly impaired formation and action of NO. Thus, replenishment of this molecule or intracellular cyclic GMP is expected so far to be the most promising therapeutic measures for patients with ED. This article includes recent advances in research on physiological roles and pathophysiological implications of NO in penile erection and on novel therapy for ED in reference to NO.

Pharmacol Ther. 2005 May;106(2):233-66

Ocular safety in patients using sildenafil citrate therapy for erectile dysfunction.

Sildenafil citrate improves erectile function in men with erectile dysfunction (ED) by selectively inhibiting cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which is present in all vascular tissue. Sildenafil also has a weaker inhibitory action on PDE6, located in the rod and cone photoreceptors. Modest, transient visual symptoms, typically blue tinge to vision, increased brightness of lights, and blurry vision, have been reported with sildenafil use and occur more frequently at higher doses. Visual function studies in healthy subjects and in patients with eye

disease suggest that sildenafil does not affect visual acuity, visual fields, and contrast sensitivity. Transient, mild impairment of color discrimination can occur around the time of peak plasma levels. Spontaneous postmarketing reports of visual adverse events, including nonarteritic anterior ischemic optic neuropathy (NAION), have been reported during the 7 years that sildenafil has been prescribed to more than 27 million men worldwide. However, because men with ED frequently have vascular risk factors that may also put them at increased risk for NAION, a causal relationship is difficult to establish. No consistent pattern has emerged to suggest any long-term effect of sildenafil on the retina or other structures of the eye or on the ocular circulation.

J Sex Med. 2006 Jan;3(1):12-27

Penile arteries and erection.

Alterations in the flow of blood to and from the penis are thought to be the most frequent causes of male erectile dysfunction and, therefore, the present review focuses on the penile vasculature. In the flaccid state, tonic noradrenaline release from the sympathetic nerves contracts penile arterial and corporal smooth muscle through activation of postjunctional alpha(1)-adrenoceptors, both by increasing intracellular calcium and by enhancing the sensitivity of the contractile apparatus for calcium. In addition, noradrenaline inhibits vasodilatatory neurotransmitter release by prejunctional alpha(2)-adrenoceptors. The exact role of the sympathetic neurotransmitters, neuropeptide Y and adenosine 5’-triphosphate, in erection is largely unknown. Penile vasodilatation during erection is mediated by nitric oxide (NO) through activation of guanylyl cyclase in the smooth muscle layer, followed by increases in cyclic guanosine monophosphate lowering of intracellular calcium and desensitisation of the contractile apparatus for calcium. Acetylcholine, vasoactive intestinal peptide as well as peptides in sensory nerves probably also play a role in penile vasodilation. Increased flow through the penile arteries stimulates the endothelium leading to release of NO, prostanoids and a non-NO non-prostanoid factor, and as such enhances the vasodilatation, while the role of endothelium-derived contractile factors in penile vasoconstriction is not clear. Erectile dysfunction shares arterial risk factors with ischaemic heart disease, and diabetes, age, and hypercholesterolaemia are associated with impairment of both neurogenic and endothelium-dependent vasodilator mechanisms in corpus cavernosum. Only few studies have investigated the impact of these risk factors on the penile vasculature, although recent evidence suggests that arterial insufficiency precedes changes in corpus cavernosum leading to erectile dysfunction.

J Vasc Res. 2002 Jul-Aug;39(4):283-303

Gender influences outcome of brain injury: progesterone plays a protective role.

The contributions of gender and gonadal hormones in the cascade of events following brain injury are largely unexplored. We measured cerebral edema following cerebral contusion in rats under three hormonal conditions to address this issue. Normally cycling females exhibited significantly less edema than males, and pseudopregnant females were virtually spared from post-injury edema. Subsequent studies of ovariectomized females, with or without hormone treatment, indicated that the reduction of cerebral edema was associated primarily with the presence of circulating progesterone. We conclude that progesterone has a protective effect on the brain following traumatic injury.

Brain Res. 1993 Apr 2;607(1-2):333-6

Progesterone facilitates cognitive recovery and reduces secondary neuronal loss caused by cortical contusion injury in male rats.

The ability of progesterone to reduce the cerebral edema associated with traumatic brain damage first became apparent when we observed that males had significantly more edema than females after cortical contusion. In addition, edema was almost absent in pseudopregnant female rats, a condition in which progesterone levels are high relative to estrogen. Progesterone injections given after injury also reduced edema and were equally effective in both males and females. The present experiment was done to determine if the progesterone-induced reduction in edema could also prevent secondary neuronal degeneration and reduce the behavioral impairments that accompany contusion of the medial frontal cortex. Progesterone-treated rats were less impaired on a Morris water maze spatial navigation task than rats treated with the oil vehicle. Progesterone-treated rats also showed less neuronal degeneration 21 days after injury in the medial dorsal thalamic nucleus, a structure that has reciprocal connections with the contused area.

Exp Neurol. 1994 Sep;129(1):64-9

Progesterone rapidly decreases brain edema: treatment delayed up to 24 hours is still effective.

Cerebral edema is a serious side effect of traumatic brain injury. We have previously established that progesterone injections, initiated within 1 h after cortical contusion injury, reduced edema when assessed 3 days later. To determine how rapidly progesterone can reduce edema, male and female rats were given the hormone 1 h after damage to the medial frontal cortex, and edema levels were assessed between 2 h and 7 days postinjury. Progesterone decreased edema with 6 h of the injury and continued to be effective for the duration of treatment. In addition, we assessed whether progesterone injections are effective when delays are imposed between injury and initiation of treatment. Male and female rats received progesterone after postinjury delays 6, 24, or 48 h. Progesterone was effective in reducing edema when treatment was delayed until 24 h after injury.

Exp Neurol. 1996 Apr;138(2):246-51

ProTECT: a randomized clinical trial of progesterone for acute traumatic brain injury.

STUDY OBJECTIVE: Laboratory evidence indicates that progesterone has potent neuroprotective effects. We conducted a pilot clinical trial to assess the safety and potential benefit of administering progesterone to patients with acute traumatic brain injury. METHODS: This phase II, randomized, double-blind, placebo-controlled trial was conducted at an urban Level I trauma center. One hundred adult trauma patients who arrived within 11 hours of injury with a postresuscitation Glasgow Coma Scale score of 4 to 12 were enrolled with proxy consent. Subjects were randomized on a 4:1 basis to receive either intravenous progesterone or placebo. Blinded observers assessed patients daily for the occurrence of adverse events and signs of recovery. Neurologic outcome was assessed 30 days postinjury. The primary safety measures were differences in adverse event rates and 30-day mortality. The primary measure of benefit was the dichotomized Glasgow Outcome Scale-Extended 30 days postinjury. RESULTS: Seventy-seven patients received progesterone; 23 received placebo. The groups had similar demographic and clinical characteristics. Laboratory and physiologic characteristics were similar at enrollment and throughout treatment. No serious adverse events were attributed to progesterone. Adverse and serious adverse event rates were similar in both groups, except that patients randomized to progesterone had a lower 30-day mortality rate than controls (rate ratio 0.43; 95% confidence interval 0.18 to 0.99). Thirty days postinjury, the majority of severe traumatic brain injury survivors in both groups had relatively poor Glasgow Outcome Scale-Extended and Disability Rating Scale scores. However, moderate traumatic brain injury survivors who received progesterone were more likely to have a moderate to good outcome than those randomized to placebo. CONCLUSION: In this small study, progesterone caused no discernible harm and showed possible signs of benefit.

Ann Emerg Med. 2007 Apr;49(4):391-402

Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury: a randomized controlled trial.

BACKGROUND: Severe traumatic brain injury (TBI) has been increasing with greater incidence of injuries from traffic or sporting accidents. Although there are a number of animal models of TBI using progesterone for head injury, the effects of progesterone on neurologic outcome of acute TBI patients remain unclear. The aim of the present clinical study was to assess the longer-term efficacy of progesterone on the improvement in neurologic outcome of patients with acute severe TBI. METHODS: A total of 159 patients who arrived within 8 hours of injury with a Glasgow Coma Score </= 8 were enrolled in the study. A prospective, randomized, placebo-controlled trial of progesterone was conducted in the Neurotrauma Center of our teaching hospital. The patients were randomized to receive either progesterone or placebo. The primary endpoint was the Glasgow Outcome Scale score 3 months after brain injury. Secondary efficacy endpoints included the modified Functional Independence Measure score and mortality. In a follow-up protocol at 6 months, the Glasgow Outcome Scale and the modified Functional Independence Measure scores were again determined. RESULTS: Of the 159 patients randomized, 82 received progesterone and 77 received placebo. The demographic characteristics, the mechanism of injury, and the time of treatment were compared for the two groups. After 3 months and 6 months of treatment, the dichotomized Glasgow Outcome Scale score analysis exhibited more favorable outcomes among the patients who were given progesterone compared with the control individuals (P = 0.034 and P = 0.048, respectively). The modified Functional Independence Measure scores in the progesterone group were higher than those in the placebo group at both 3-month and 6-month follow-up (P < 0.05 and P < 0.01). The mortality rate of the progesterone group was significantly lower than that of the placebo group at 6-month follow-up (P < 0.05). The mean intracranial pressure values 72 hours and 7 days after injury were lower in the progesterone group than in the placebo group, but there was no statistical significance between the two groups (P > 0.05). Instances of complications and adverse events associated with the administration of progesterone were not found. CONCLUSION: Our data suggest that acute severe TBI patients with administration of progesterone hold improved neurologic outcomes for up to 6 months. These results provide information important for further large and multicenter clinical trials on progesterone as a promising neuroprotective drug.

Crit Care. 2008;12(2):R61

Progesterone exerts neuroprotective effects after brain injury.

Progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. This review assesses recent, primarily in vivo, evidence that progesterone can play an important role in promoting and enhancing repair after traumatic brain injury and stroke. Although many of its specific actions on neuroplasticity remain to be discovered, there is growing evidence that this hormone may be a safe and effective treatment for traumatic brain injury and other neural disorders in humans.

Brain Res Rev. 2008 Mar;57(2):386-97

The membrane-associated progesterone-binding protein 25-Dx: expression, cellular localization and up-regulation after brain and spinal cord injuries.

Progesterone has neuroprotective effects in the injured and diseased spinal cord and after traumatic brain injury (TBI). In addition to intracellular progesterone receptors (PR), membrane-binding sites of progesterone may be involved in neuroprotection. A first putative membrane receptor of progesterone, distinct from the classical intracellular PR isoforms, with a single membrane-spanning domain, has been cloned from porcine liver. Homologous proteins were cloned in rats (25-Dx), mice (PGRMC1) and humans (Hpr.6). We will refer to this progesterone-binding protein as 25-Dx. The distribution and regulation of 25-Dx in the nervous system may provide some clues to its functions. In spinal cord, 25-Dx is localized in cell membranes of dorsal horn neurons and ependymal cells lining the central canal. A role of 25-Dx in mediating the protective effects of progesterone in the spinal cord is supported by the observation that its mRNA and protein are up-regulated by progesterone in dorsal horn of the injured spinal cord. In contrast, the classical intracellular PRs were down-regulated under these conditions. In brain, 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, ependymal cells of the ventricular walls, and the meninges. Interestingly, it is co-expressed with vasopressin in neurons of the paraventricular, supraoptic and retrochiasmatic nuclei. In response to TBI, 25-Dx expression is up-regulated in neurons and induced in astrocytes. The expression of 25-Dx in structures involved in cerebrospinal fluid production and osmoregulation, and its up-regulation after brain damage, point to a potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after TBI. Our observations suggest that progesterone’s actions may involve different signaling mechanisms depending on the pathophysiological context, and that 25-Dx may be involved in the neuroprotective effect of progesterone in the injured brain and spinal cord.

Brain Res Rev. 2008 Mar;57(2):493-505

Progesterone: therapeutic opportunities for neuroprotection and myelin repair.

Progesterone and its metabolites promote the viability of neurons in the brain and spinal cord. Their neuroprotective effects have been documented in different lesion models, including traumatic brain injury (TBI), experimentally induced ischemia, spinal cord lesions and a genetic model of motoneuron disease. Progesterone plays an important role in developmental myelination and in myelin repair, and the aging nervous system appears to remain sensitive to some of progesterone’s beneficial effects. Thus, the hormone may promote neuroregeneration by several different actions by reducing inflammation, swelling and apoptosis, thereby increasing the survival of neurons, and by promoting the formation of new myelin sheaths. Recognition of the important pleiotropic effects of progesterone opens novel perspectives for the treatment of brain lesions and diseases of the nervous system. Over the last decade, there have been a growing number of studies showing that exogenous administration of progesterone or some of its metabolites can be successfully used to treat traumatic brain and spinal cord injury, as well as ischemic stroke. Progesterone can also be synthesized by neurons and by glial cells within the nervous system. This finding opens the way for a promising therapeutic strategy, the use of pharmacological agents, such as ligands of the translocator protein (18 kDa) (TSPO; the former peripheral benzodiazepine receptor or PBR), to locally increase the synthesis of steroids with neuroprotective and neuroregenerative properties. A concept is emerging that progesterone may exert different actions and use different signaling mechanisms in normal and injured neural tissue.

Pharmacol Ther. 2007 Oct;116(1):77-106

Does progesterone have neuroprotective properties?

In this article, we review published preclinical and epidemiologic studies that examine progesterone’s role in the central nervous system. Its effects on the reproductive and endocrine systems are well known, but a large and growing body of evidence, including a recently published pilot clinical trial, indicates that the hormone also exerts neuroprotective effects on the central nervous system. We now know that it is produced in the brain, for the brain, by neurons and glial cells in the central and peripheral nervous system of both male and female individuals. Laboratories around the world have reported that administering relatively large doses of progesterone during the first few hours to days after injury significantly limits central nervous system damage, reduces loss of neural tissue, and improves functional recovery. Although the research published to date has focused primarily on progesterone’s effects on blunt traumatic brain injury, there is evidence that the hormone affords protection from several forms of acute central nervous system injury, including penetrating brain trauma, stroke, anoxic brain injury, and spinal cord injury. Progesterone appears to exert its protective effects by protecting or rebuilding the blood-brain barrier, decreasing development of cerebral edema, down-regulating the inflammatory cascade, and limiting cellular necrosis and apoptosis. All are plausible mechanisms of neuroprotection.

Ann Emerg Med. 2008 Feb;51(2):164-72

Progesterone improves acute recovery after traumatic brain injury in the aged rat.

Recent evidence has demonstrated that treatment with progesterone can attenuate many of the pathophysiological events following traumatic brain injury (TBI) in young adult rats, but this effect has not been investigated in aged animals. In this study, 20-month-old male Fischer 344 rats with bilateral contusions of the frontal cortex (n = 4 per group) or sham operations received 8, 16, or 32 mg/kg of progesterone or vehicle. Locomotor activity was measured at 72 h to assess behavioral recovery. Brain tissue was harvested at 24, 48, and 72 h, and Western blotting was performed for inflammatory and apoptotic factors. Edema was assessed at 48 h by measuring brain water content. Injured animals treated with 8 and 16 mg/kg progesterone showed decreased expression of COX-2, IL-6, and NFkappaB at all time points, indicating a reduction in the acute inflammatory process compared to vehicle. The 16 mg/kg group also showed reduced apoptosis at all time points as well as decreased edema and improved locomotor outcomes. Thus, in aged male rats, treatment with 16 mg/kg progesterone improves short-term motor recovery and attenuates edema, secondary inflammation, and cell death after TBI.

J Neurotrauma. 2007 Sep;24(9):1475-86

Ellagic acid, pomegranate and prostate cancer — a mini review.

There is currently a shifting focus towards finding natural compounds that may prevent or treat cancer, due to the problems that exist with current chemotherapeutic regimens. The fruit of the Punica granatum (pomegranate) contains hundreds of phytochemicals and pomegranate extracts have recently been shown to exhibit antioxidant properties, thought to be due to the action of ellagic acid, the main polyphenol in pomegranate. In this mini review the effects of pomegranate extracts and ellagic acid on the proliferation of prostate cancer cells and their future potential are discussed.

J Pharm Pharmacol. 2008 Feb;60(2):139-44

Therapeutic applications of pomegranate (Punica granatum L.): a review.

The pomegranate, Punica granatum L., is an ancient, mystical, unique fruit borne on a small, long-living tree cultivated throughout the Mediterranean region, as far north as the Himalayas, in Southeast Asia, and in California and Arizona in the United States. In addition to its ancient historical uses, pomegranate is used in several systems of medicine for a variety of ailments. The synergistic action of the pomegranate constituents appears to be superior to that of single constituents. In the past decade, numerous studies on the antioxidant, anticarcinogenic, and anti-inflammatory properties of pomegranate constituents have been published, focusing on treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, bacterial infections and antibiotic resistance, and ultraviolet radiation-induced skin damage. Other potential applications include infant brain ischemia, male infertility, Alzheimer’s disease, arthritis, and obesity.

Altern Med Rev. 2008 Jun;13(2):128-44

Pomegranate juice: a heart-healthy fruit juice.

Pomegranate juice is a polyphenol-rich fruit juice with high antioxidant capacity. In limited studies in human and murine models, pomegranate juice has been shown to exert significant antiatherogenic, antioxidant, antihypertensive, and anti-inflammatory effects. Pomegranate juice significantly reduced atherosclerotic lesion areas in immune-deficient mice and intima media thickness in cardiac patients on medications. It also decreased lipid peroxidation in patients with type 2 diabetes, and systolic blood pressure and serum angiotensin converting enzyme activity in hypertensive patients. Thus, the potential cardioprotective benefits of pomegranate juice deserve further clinical investigation, and evidence to date suggests it may be prudent to include this fruit juice in a heart-healthy diet.

Nutr Rev. 2009 Jan;67(1):49-56

Pomegranate derived products for cancer chemoprevention.

Because treatment options for advanced metastasized cancers remain inadequate, developing effective approaches for the prevention of cancer has become an important goal to reduce cancer burden. One such strategy is through chemoprevention, preferably by the use of non-toxic dietary substances and botanical products. Pomegranate, used for centuries for its medicinal properties is now being recognized as a potential chemopreventive and anticancer agent. Increasing body of evidence has underscored the cancer preventive efficacy of pomegranate both in vitro and in vivo animal models. The emerging data provide new insights into the molecular framework needed to establish novel mechanism-based chemopreventive strategies for various human cancers.

Semin Cancer Biol. 2007 Oct;17(5):377-85

Pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis.

Inhibition of lipid peroxidation contributes to the attenuation of macrophage cholesterol accumulation, foam-cell formation and atherosclerosis. Evidence suggests that nutritional antioxidants such as pomegranate juice (PJ) can contribute to the reduction of oxidative stress and atherogenesis. The goals of the present study were to determine whether such beneficial effects of PJ exist when supplemented to apolipoprotein E-deficient (E(0)) mice with advanced atherosclerosis and to analyze the antiatherosclerotic activity of a tannin-fraction isolated from PJ. Mice (4-mo-old) were supplemented with PJ in their drinking water for 2 mo and compared with age-matched placebo-treated mice, as well as to young (4-mo-old) control mice, for their mouse peritoneal macrophage (MPM) oxidative state, cholesterol flux and mice atherosclerotic lesion size. PJ supplementation reduced each of the proatherogenic variables determined in the present study compared with age-matched placebo-treated mice. It significantly induced serum paraoxonase activity and reduced MPM lipid peroxide content compared with placebo-treated mice and control mice. PJ administration to E(0) mice significantly reduced the oxidized (Ox)-LDL MPM uptake by 31% and MPM cholesterol esterification and increased macrophage cholesterol efflux by 39% compared with age-matched, placebo-treated mice. PJ consumption reduced macrophage Ox-LDL uptake and cholesterol esterification to levels lower than those in 4-mo-old, unsupplemented controls. PJ supplementation to E(0) mice with advanced atherosclerosis reduced the lesion size by 17% compared with placebo-treated mice. In a separate study, supplementation of young (2-mo-old) E(0) mice for 2 mo with a tannin fraction isolated from PJ reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased Ox-LDL MPM uptake. PJ supplementation to mice with advanced atherosclerosis reduced their macrophage oxidative stress, their macrophage cholesterol flux and even attenuated the development of atherosclerosis. Moreover, a tannin-fraction isolated from PJ had a significant antiatherosclerotic activity.

J Nutr. 2001 Aug;131(8):2082-9

Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice.

BACKGROUND: Dietary supplementation with nutrients rich in antioxidants is associated with inhibition of atherogenic modifications to LDL, macrophage foam cell formation, and atherosclerosis. Pomegranates are a source of polyphenols and other antioxidants. OBJECTIVE: We analyzed, in healthy male volunteers and in atherosclerotic apolipoprotein E-deficient (E(0)) mice, the effect of pomegranate juice consumption on lipoprotein oxidation, aggregation, and retention; macrophage atherogenicity; platelet aggregation; and atherosclerosis. DESIGN: Potent antioxidative effects of pomegranate juice against lipid peroxidation in whole plasma and in isolated lipoproteins (HDL and LDL) were assessed in humans and in E(0) mice after pomegranate juice consumption for </=2 and 14 wk, respectively. RESULTS: In humans, pomegranate juice consumption decreased LDL susceptibility to aggregation and retention and increased the activity of serum paraoxonase (an HDL-associated esterase that can protect against lipid peroxidation) by 20%. In E(0) mice, oxidation of LDL by peritoneal macrophages was reduced by up to 90% after pomegranate juice consumption and this effect was associated with reduced cellular lipid peroxidation and superoxide release. The uptake of oxidized LDL and native LDL by mouse peritoneal macrophages obtained after pomegranate juice administration was reduced by 20%. Finally, pomegranate juice supplementation of E(0) mice reduced the size of their atherosclerotic lesions by 44% and also the number of foam cells compared with control E(0) mice supplemented with water. CONCLUSION: Pomegranate juice had potent antiatherogenic effects in healthy humans and in atherosclerotic mice that may be attributable to its antioxidative properties.

Am J Clin Nutr. 2000 May;71(5):1062-76

Pomegranate juice flavonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans.

The beneficial health effects attributed to the consumption of fruit and vegetables are related, at least in part, to their antioxidant activity. Of special interest is the inverse relationship between the intake of dietary nutrients rich in polyphenols and cardiovascular diseases. This effect is attributed to polyphenols’ ability to inhibit low-density lipoprotein (LDL) oxidation, macrophage foam cell formation and atherosclerosis. Pomegranate polyphenols can protect LDL against cell-mediated oxidation via two pathways, including either direct interaction of the polyphenols with the lipoprotein and/or an indirect effect through accumulation of polyphenols in arterial macrophages. Pomegranate polyphenols were shown to reduce the capacity of macrophages to oxidatively modify LDL, due to their interaction with LDL to inhibit its oxidation by scavenging reactive oxygen species and reactive nitrogen species and also due to accumulation of polyphenols in arterial macrophages; hence, the inhibition of macrophage lipid peroxidation and the formation of lipid peroxide-rich macrophages. Furthermore, pomegranate polyphenols increase serum paraoxonase activity, resulting in the hydrolysis of lipid peroxides in oxidized lipoproteins and in atherosclerotic lesions. These antioxidative and antiatherogenic effects of pomegranate polyphenols were demonstrated in vitro, as well as in vivo in humans and in atherosclerotic apolipoprotein E deficient mice. Dietary supplementation of polyphenol-rich pomegranate juice to atherosclerotic mice significantly inhibited the development of atherosclerotic lesions and this may be attributed to the protection of LDL against oxidation.

Drugs Exp Clin Res. 2002;28(2-3):49-62

Pomegranate byproduct administration to apolipoprotein e-deficient mice attenuates atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized low-density lipoprotein.

The effects of a pomegranate byproduct (PBP, which includes the whole pomegranate fruit left after juice preparation) on atherosclerosis development in apolipoprotein E-deficient (E degrees ) mice were studied. Consumption of PBP (17 or 51.5 microg of gallic acid equiv/kg/day) by the mice resulted in a significant reduction in atherosclerotic lesion size by up to 57%. PBP consumption significantly reduced oxidative stress in the mice peritoneal macrophages (MPM): Cellular lipid peroxide content decreased by up to 42%, the reduced glutathione levels increased by up to 53%, and paraoxonase 2 lactonase activity increased by up to 50%, as compared to MPM from E degrees mice that consumed only water. Furthermore, oxidized low-density lipoprotein (Ox-LDL) uptake by the MPM was reduced by up to 19%. Similar results were observed also in vitro. Treatment of J774A.1 macrophages with PBP (10 or 50 micromol/L of total polyphenols) significantly decreased both cellular total peroxide content and Ox-LDL uptake. It was thus concluded that PBP significantly attenuates atherosclerosis development by its antioxidant properties.

J Agric Food Chem. 2006 Mar 8;54(5):1928-35

Pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure.

Consumption of pomegranate juice which is rich in tannins, possess anti-atherosclerotic properties which could be related to its potent anti-oxidative characteristics. As some antioxidants were recently shown to reduce blood pressure, we studied the effect of pomegranate juice consumption (50 ml, 1.5mmol of total polyphenols per day, for 2 weeks) by hypertensive patients on their blood pressure and on serum angiotensin converting enzyme (ACE) activity. A 36% decrement in serum ACE activity and a 5% reduction in systolic blood pressure were noted. Similar dose-dependent inhibitory effect (31%) of pomegranate juice on serum ACE activity was observed also in vitro. As reduction in serum ACE activity, even with no decrement in blood pressure, was previously shown to attenuate atherosclerosis, pomegranate juice can offer a wide protection against cardiovascular diseases which could be related to its inhibitory effect on oxidative stress and on serum ACE activity.

Atherosclerosis. 2001 Sep;158(1):195-8

Pomegranate juice consumption for 3 years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and LDL oxidation.

Dietary supplementation with polyphenolic antioxidants to animals was shown to be associated with inhibition of LDL oxidation and macrophage foam cell formation, and attenuation of atherosclerosis development. We investigated the effects of pomegranate juice (PJ, which contains potent tannins and anthocyanins) consumption by atherosclerotic patients with carotid artery stenosis (CAS) on the progression of carotid lesions and changes in oxidative stress and blood pressure. Ten patients were supplemented with PJ for 1 year and five of them continued for up to 3 years. Blood samples were collected before treatment and during PJ consumption. In the control group that did not consume PJ, common carotid intima-media thickness (IMT) increased by 9% during 1 year, whereas, PJ consumption resulted in a significant IMT reduction, by up to 30%, after 1 year. The patients’ serum paraoxonase 1 (PON 1) activity was increased by 83%, whereas serum LDL basal oxidative state and LDL susceptibility to copper ion-induced oxidation were both significantly reduced, by 90% and 59%, respectively, after 12 months of PJ consumption, compared to values obtained before PJ consumption. Furthermore, serum levels of antibodies against oxidized LDL were decreased by 19%, and in parallel serum total antioxidant status (TAS) was increased by 130% after 1 year of PJ consumption.

Systolic blood pressure was reduced after 1 year of PJ consumption by 12% [corrected] and was not further reduced along 3 years of PJ consumption. For all studied parameters, the maximal effects were observed after 1 year of PJ consumption. Further consumption of PJ, for up to 3 years, had no additional beneficial effects on IMT and serum PON1 activity, whereas serum lipid peroxidation was further reduced by up to 16% after 3 years of PJ consumption. The results of the present study thus suggest that PJ consumption by patients with CAS decreases carotid IMT and systolic blood pressure and these effects could be related to the potent antioxidant characteristics of PJ polyphenols.

Clin Nutr. 2004 Jun;23(3):423-33

Consumption of wonderful variety pomegranate juice and extract by diabetic patients increases paraoxonase 1 association with high-density lipoprotein and stimulates its catalytic activities.

Association of paraoxonase 1 (PON1) with high-density lipoprotein (HDL) stabilizes the enzyme. In diabetic patients, PON1 dissociates from HDL and, as a consequence, is less biologically active. Our aim was to investigate the effects of Wonderful variety pomegranate juice (WPJ) and pomegranate polyphenol extract (WPOMxl) consumption on PON1 association with HDL in diabetic patients. Thirty patients with type 2 diabetes mellitus participated in the study. Ten male patients and 10 female patients received concentrated WPJ (50 mL/day for 4 weeks), while another group of 10 male patients received WPOMxl (5 mL/day for 6 weeks). There were no significant effects of WPJ or WPOMxl consumption on fasting blood glucose or hemoglobin A1c levels. After 4 weeks of WPJ consumption by male patients, basal serum oxidative stress was significantly decreased by 35%, whereas serum concentrations of thiol groups significantly increased by 25%. Moreover, HDL-associated PON1 arylesterase, paraoxonase, and lactonase activities increased significantly after WPJ consumption by 34-45%, as compared to the baseline levels. PON1 protein binding to HDL was significantly increased by 30% following WPJ consumption, and the enzyme became more stable. In male patients that consumed WPOMxl and in female patients that consumed PJ, a similar pattern was observed, although to a lesser extent. We conclude that WPJ as well as WPOMxl consumption by diabetic patients does not worsen their diabetic parameters. Furthermore, WPJ as well as WPOMxl consumption contribute to PON1 stabilization, increased association with HDL, and enhanced catalytic activities. These beneficial effects of pomegranate consumption on serum PON1 stability and activity could lead to retardation of atherosclerosis development in diabetic patients.

J Agric Food Chem. 2008 Sep 24;56(18):8704-13

Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPAR gamma and AP-1 pathway activation.

Paraoxonase 2 (PON2), a member of the paraoxonase gene family, was shown to protect macrophages against oxidative stress. Pomegranate juice (PJ), which contains potent polyphenol anti-oxidants, exhibits similar effects. We questioned possible association between PJ polyphenolics, macrophage oxidative stress, and cellular PON2 expression, in relation to the activation of specific PON2 transcription factors. Incubation of J774A.1 macrophages with PJ (0-50 microM of total polyphenols) dose-dependently increased expression (mRNA, protein) and activity and reduced macrophage oxidative status. These effects could be attributed to the PJ unique polyphenols, punicalagin and gallic acid. PJ polyphenol-induced up-regulation of PON2 was inhibited by 40% upon using the PPAR gamma inhibitor GW9662 (50 microM). Accordingly, the PPAR gamma ligand, rosiglitazone, dose-dependently stimulated macrophage PON2 expression, by up to 80%. Inhibition of AP-1 activation with SP600125, attenuated PJ-induced up-regulation of PON2 by 40%. Similarly, incubation of macrophages with PJ polyphenols in the presence of GW9662 or SP600125, significantly reduced their capacity to protect the cells against oxidative stress. We conclude that the anti-oxidative characteristics of PJ unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage PON2 expression, a phenomenon which was shown to be associated with activation of the transcription factors PAPR gamma and AP-1.

Atherosclerosis. 2007 Dec;195(2):313-21

Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules.

Glycation is a slow chemical reaction which takes place between amino residues in protein and a reducing sugar. In skin this reaction creates new residues or induces the formation of cross-links (advanced glycation end products or AGEs) in the extracellular matrix of the dermis. Formation of such cross-links between macromolecules may be responsible for loss of elasticity or modification of other properties of the dermis observed during aging. We had previously developed a reconstructed skin model which enabled us to study the consequences of matrix alteration by preglycation of the collagen and have reported several modifications of interest induced by glycation in the dermal and epidermal compartments of reconstructed skin as well as at the level of the dermal-epidermal junction. For example we showed that collagen IV and laminin were increased in the basement membrane zone and that alpha6 and beta1 integrins in epidermis were expanded to suprabasal layers. The aim of this new study was to look at the biological effects of glycation inhibitors like aminoguanidine in the skin model. Aminoguanidine was mixed with collagen in the presence of ribose as reducing sugar, and immunostaining was used to visualize its effects on AGE Products and biological markers. After aminoguanidine treatment, we found a low amount of AGE products and a possible return to the normal pattern of distribution of markers in skin constructs as compared to those treated with ribose only. Interestingly similar results were also obtained, although to a lesser extent, with a blueberry extract. In conclusion the glycation inhibitory effect has been functionally demonstrated in the reconstructed skin model and it is shown that this model can be used to assess anti-glycation agents.

Exp Gerontol. 2008 Jun;43(6):584-8

Glycation—a sweet tempter for neuronal death.

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Brain Res Brain Res Rev. 2003 Mar;41(2-3):306-23

The receptor for advanced glycation end products is highly expressed in the skin and upregulated by advanced glycation end products and tumor necrosis factor-alpha.

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, factor XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and keratinocytes and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nepsilon-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis factor-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue growth factor, transforming growth factor-beta (TGF-beta) 1, and procollagen-alpha1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, -2, -3, and -12 was unaffected. We conclude that in HFFs, AGE-RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression.

J Invest Dermatol. 2006 Feb;126(2):291-9

Effect of advanced glycation end-products on cell proliferation and cell death.

The effect of advanced glycation end products (AGE-s) was studied on the proliferation and cell death of human skin fibroblasts in culture. Several AGE-products were prepared from proteins, a peptide and amino acids, using Glucose or Fructose, with or without Fe2+. The AGE preparations increased cell death at the 7th day, after only 72 hours of incubation. Some of these glycation products modified also proliferation. This effect of AGE-s was even maintained without these products in fresh medium for a second period of incubation up to 10 days from the start of the experiment. In order to explore the role of AGE-receptors, especially of AGE-receptor and of growth factor receptors (fibroblast and epidermal growth factors receptors), antibodies to these receptors were added to cell cultures and their effect on both cell death and proliferation were determined as for the AGE-s. These anti-receptor antibodies imitated to some extent the results obtained with AGE-s, producing increase of cell death and proliferation, followed above a certain concentration of antibodies by a decrease and a new increase or plateau. This might correspond to the internalization of the receptors followed by a re-expression on the cell membrane. The role of receptor-mediated Reactive Oxygen Species-production was also explored using scavengers: N-acetyl-cysteine (NAC), L-Carnosine, superoxide dismutase (SOD) and Catalase. Several of these scavengers decreased cell death, suggesting that Reactive Oxygen Species-production is partially involved in the observed phenomena.

Pathol Biol (Paris). 2006 Sep;54(7):396-404

Glycation induces expansion of the molecular packing of collagen.

Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.

J Mol Biol. 1988 Sep 20;203(2):495-505

Advanced glycation end products induce crosslinking of collagen in vitro.

We have investigated the effect of advanced glycation end products (AGEs) on the crosslinking of collagen. The potential pathological significance of AGEs and the altered metabolism of ascorbic acid (ASA) in diabetes have prompted us to investigate the role of ASA in the crosslinking and advanced glycation of collagen. Rat tail tendons were incubated with ASA and dehydroascorbic acid (DHA) under physiological conditions of temperature and pH, and the crosslinking and the level of AGEs were analyzed. Analysis of crosslinking was conducted by pepsin solubility and cyanogen bromide digestion. Level of AGEs was estimated by enzyme-linked immunosorbent assay (ELISA) using antibodies raised against AGE-ribonuclease. It was noted that ASA and DHA induced crosslinking of collagen and stimulated the formation of AGEs. It was also noted that these pathways were dependent on oxidative conditions. Similarly incubation of collagen with AGEs, prepared by the in vitro incubation of bovine serum albumin (BSA) with glucose, also resulted in increased crosslinking. The extent of crosslinking was dependent on the duration of incubation. The novel finding of this study, which is in contrast to the earlier reports on glucose-induced crosslinking of collagen, was that AGEs-induced crosslinking of collagen was not inhibited by radical scavengers and the metal chelator. EDTA, whereas glucose-induced crosslinking of collagen was almost completely prevented by free radical scavengers. The increased fluorescence intensity observed in collagen incubated with AGEs was also not prevented by radical scavengers. Estimation of AGEs by ELISA revealed an increased accumulation of AGEs in collagen incubated with AGE-BSA. The inhibitory effect of aminoguanidine and aspirin on AGEs-induced modification of collagen, strongly suggests that the amino-carbonyl interaction between AGEs and collagen may play a key role in the crosslinking process. The results obtained in this study indicate that soluble AGEs can directly induce crosslinking of collagen and this process is independent of oxidative conditions. From these results it may be hypothesized that glucose, under oxidative conditions, reacts with proteins to form potentially reactive end products called AGEs. These AGEs, once formed, could induce crosslinking of collagen even in the absence of both glucose and oxygen.

Biochim Biophys Acta. 1998 Sep 30;1407(3):215-24

Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways.

Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-epsilon-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor alpha.

J Biol Chem. 2005 Apr 1;280(13):12087-95

Decrease in skin collagen glycation with improved glycemic control in patients with insulin-dependent diabetes mellitus.

Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions.

J Clin Invest. 1991 Jun;87(6):1910-5

Dysfunction of dermal fibroblasts induced by advanced glycation end-products (AGEs) and the contribution of a nonspecific interaction with cell membrane and AGEs.

Advanced glycation end-products (AGEs) have been reported to accumulate in the dermal skin. However, it remains unknown whether the AGEs interact with the dermal fibroblasts and influence their function. Previously, we demonstrated that AGEs hastened photoaging of the skin by means of active oxygen species such as *O(2)(-), H(2)O(2), and *OH, generated during UVA irradiation. The purpose of the present study was to clarify the influence of AGEs on the functions of dermal fibroblasts under physiological conditions. It was found that AGEs decreased both hyaluronic acid (HA) synthesis and activity of elastase-type matrix metalloproteinase (ET-MMP). Because the reactions of both HA synthesis and ET-MMP were found to take place at the cell membrane region, it appeared that AGEs modulated cellular dysfunction through an interaction with the cell membrane. To clarify the mechanisms of these dysfunction in relation to AGEs, we examined the interaction between AGEs and cell membranes, and obtained the following results: (1) AGEs associated with the cell membranes and liposomal membrane prepared with phosphatidyl choline; (2) AGEs hydrophobically modified the circumstances of the cell membrane and liposome membrane as evaluated by experiments using a fluorescence probe; (3) AGEs increased the fluidity of the cell membrane and liposomal membrane as estimated by ESR spin-labeling using 5-doxylstearic acid; and (4) AGEs accelerated lactate dehydrogenase (LDH) leakage from the cells. On the basis of these experimental results, we proposed that AGEs modulated cell function through a nonspecific interaction with the membranes of dermal fibroblasts.

J Dermatol Sci. 2002 Sep;29(3):171-80