Life Extension Magazine®

Issue: Sep 2015

Taurine, Endothelial Dysfunction, Fucoidan, and Thymus Regeneration

Taurine, Endothelial Dysfunction, Fucoidan, and Thymus Regeneration

By Life Extension.

Taurine

Taurine increases hippocampal neurogenesis in aging mice.

Aging is associated with increased inflammation and reduced hippocampal neurogenesis, which may in turn contribute to cognitive impairment. Taurine is a free amino acid found in numerous diets, with anti-inflammatory properties. Although abundant in the young brain, the decrease in taurine concentration with age may underlie reduced neurogenesis. Here, we assessed the effect of taurine on hippocampal neurogenesis in middle-aged mice. We found that taurine increased cell proliferation in the dentate gyrus through the activation of quiescent stem cells, resulting in increased number of stem cells and intermediate neural progenitors. Taurine had a direct effect on stem/progenitor cells proliferation, as observed in vitro, and also reduced activated microglia. Furthermore, taurine increased the survival of newborn neurons, resulting in a net increase in adult neurogenesis. Together, these results show that taurine increases several steps of adult neurogenesis and support a beneficial role of taurine on hippocampal neurogenesis in the context of brain aging.

Stem Cell Res. 2015 May;14(3):369-79

Antenatal taurine improves neuronal regeneration in fetal rats with intrauterine growth restriction by inhibiting the Rho-ROCK signal pathway.

The Rho-ROCK signal pathway is an important mediator of inhibitory signals that blocks central nervous cell regeneration. Here, we investigated whether antenatal taurine improved neuronal regeneration in fetal rats with intrauterine growth restriction (IUGR) by inhibiting this pathway. Thirty pregnant rats were randomly divided into three groups: control, IUGR, and IUGR + antenatal taurine supplementation (taurine group). The mRNA levels of Ras homolog gene A (Rho A), Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and proliferating cell nuclear antigen (PCNA) were detected using real-time quantitative PCR. RhoA, ROCK2 and PCNA-positive cells were counted using immunohistochemistry. Antenatal taurine supplementation decreased RhoA and Rock2 mRNA expression, increased PCNA mRNA expression, and significantly decreased RhoA, ROCK2-positive and increased PCNA-positive cell counts in IUGR fetal rat brain tissues (p < 0.05). Thus, antenatal taurine supplementation inhibited the expression of key Rho-ROCK signal molecules and improved IUGR fetal brain development.

Metab Brain Dis . 2015 Feb;30(1):67-73

Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Dev Neurosci. 2013;35(1):40-9

Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer’s disease.

Alzheimer’s disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Ab. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Ab-related damages.

Sci Rep . 2014 Dec 12;4:7467

Therapeutic effect of taurine against aluminum-induced impairment on learning, memory and brain neurotransmitters in rats.

The aim of the study was to demonstrate the therapeutic effect of taurine against aluminum (Al)-induced neurological disorders in rats. Forty-two Wistar rats were randomly allotted into six groups: control (saline only), Al exposure (281.4 mg/kg/day for 1 month), Al + taurine (Al administration as previously plus taurine, doses were 200, 400, and 800 mg/kg/day, respectively, for the next 1 month) and prevention group (along with the Al administration as previously, 400 mg/kg/day taurine was treated for 1 month. During the next 1 month, rats were given taurine 400 mg/kg/day only). Starting from the sixth week, the body weight gain was significantly reduced in Al exposure group compared with saline (P < 0.05), and at the eighth week, the gain in prevention group was increased compared with Al (P < 0.05). Brain coefficient was gained in Al exposure compared with saline or prevention group (P < 0.05). Al exposure resulted in learning and memory impairment by increasing the escape latency and searching distance, meanwhile, decreasing the swimming time in the quadrant of platform and the numbers of crossing the platform (P < 0.05). Unsurprisingly, taurine treatment (400, 800 mg/kg/day and prevention) significantly protected against Al-induced brain dysfunction (P < 0.05). The Al exposure led to significant decreases in levels of g-GABA and Tau, meanwhile, increased in level of Asp and Glu compared with saline (P < 0.05). And yet, taurine treatment partially reversed the deteriorated changes. The results suggested that taurine probably has neuroprotective effect against Al-induced learning, memory and brain neurotransmitters dysfunction.

Neurol Sci. 2014 Oct;35(10):1579-84

Reduced plasma taurine level in Parkinson’s disease: association with motor severity and levodopa treatment.

PURPOSE: This study aimed to evaluate the level of taurine in plasma, and its association with the severity of motor and non-motor symptoms (NMS) and chronic levodopa treatment inParkinson’s disease (PD). PATIENTS AND METHODS: Plasma taurine level was measured in treated PD (tPD), untreated PD (ntPD) and control groups. Motor symptoms and NMS were assessed using the Unified Parkinson’s Disease Rating Scale, the short form of the McGill Pain Questionnaire, the Hamilton Depression Scale, the Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms and the Pittsburgh Sleep Quality Index. Longtime exposure to levodopa was indicated by its approximate cumulative dosage. RESULTS: The plasma taurine levels of PD patients were decreased when compared with controls and negatively associated with motor severity but not NMS. Moreover, tPD patients exhibited lower levels of plasma taurine than ntPD patients. Interestingly, plasma taurine levels negatively correlated with cumulative levodopa dosage in tPD. After controlling for potential confounders, the association between taurine and levodopa remained significant. CONCLUSION: Our study supports that taurine may play important roles in the pathophysiology of PD and the disturbances caused by chronic levodopa administration.

Int J Neurosci. 2015 May 23:1-24

Effects of taurine on contractions of human internal mammary artery: a potassium channel opening action.

OBJECTIVE: Taurine is an abundant amino acid that is widely distributed in human and animal tissues. Pharmacodynamic studies show that taurine has hypotensive and myocardial protective effects. Studies in isolated tissue baths show that taurine relaxes precontracted arteries. This study aimed to show the effects of taurine on human internal mammary artery (IMA) in vitro and to explain the mechanisms of its effects. METHODS: The response in the IMA was recorded isometrically by a force displacement transducer in isolated organ baths. Taurine (20, 40, 80 mM) was added to organ baths after precontraction with KCl (45 mM) or serotonin (5-HT, 30 µM). Taurine-induced relaxations were also tested in the presence of the cyclooxygenase inhibitor indomethacin (10 µM), the nitric oxide synthase inhibitor L-NAME (100 µM), the large conductance Ca2+-activated K+ channel inhibitor tetraethylammonium (TEA, 1 mM), the ATP-sensitive K+ channel inhibitor glibenclamide (GLI, 10 µM), the voltage-sensitive K+ channel inhibitor 4-aminopyridine (4-AP, 1 mM) and the inward rectifier K+ channel inhibitor barium chloride (BaCl2, 30 µM). RESULTS: Taurine did not affect the resting tone of IMA. However, it produced relaxation in the 5-HT and KCl -precontracted preparations. The relaxation to IMA was not affected by GLI, 4-AP, BaCl2, indomethacin and L-NAME. But, TEA inhibited taurine -induced relaxations significantly (p < 0.05). CONCLUSIONS: The preincubation of IMA with taurine antagonized KCl and 5-HT induced contractions in a concentration dependent manner, while it did not affect the resting tone. The relaxations to taurine were significantly antagonized by pretreatment with TEA. These results suggest that mechanism of vasodilator effect of taurine in IMA may be the activation of large conductance Ca2+-activated K+ channels.

Eur Rev Med Pharmacol Sci . 2015 Apr;19(8):1498-504

Taurine supplementation ameliorates glucose homeostasis, prevents insulin and glucagon hypersecretion, and controls b, a, and d-cell masses in genetic obese mice.

Taurine (Tau) regulates b-cell function and glucose homeostasis under normal and diabetic conditions. Here, we assessed the effects of Tau supplementation upon glucose homeostasis and the morphophysiology of endocrine pancreas, in leptin-deficient obese (ob) mice. From weaning until 90-day-old, C57Bl/6 and ob mice received, or not, 5% Tau in drinking water (C, CT, ob and obT). Obese mice were hyperglycemic, glucose intolerant, insulin resistant, and exhibited higher hepatic glucose output. Tau supplementation did not prevent obesity, but ameliorated glucose homeostasis in obT. Islets from ob mice presented a higher glucose-induced intracellular Ca(2+) influx, NAD(P)H production and insulin release. Furthermore, a-cells from ob islets displayed a higher oscillatory Ca(2+) profile at low glucose concentrations, in association with glucagon hypersecretion. In Tau-supplemented ob mice, insulin and glucagon secretion was attenuated, while Ca(2+) influx tended to be normalized in b-cells and Ca(2+) oscillations were increased in a-cells. Tau normalized the inhibitory action of somatostatin (SST) upon insulin release in the obT group. In these islets, expression of the glucagon, GLUT-2 and TRPM5 genes was also restored. Tau also enhanced MafA, Ngn3 and NeuroD mRNA levels in obT islets. Morphometric analysis demonstrated that the hypertrophy of ob islets tends to be normalized by Tau with reductions in islet and b-cell masses, but enhanced d-cell mass in obT. Our results indicate that Tau improves glucose homeostasis, regulating b-, a-, and d-cell morphophysiology in ob mice, indicating that Tau may be a potential therapeutic tool for the preservation of endocrine pancreatic function in obesity and diabetes.

Amino Acids . 2015 Aug;47(8):1533-48

Taurine counteracts the suppressive effect of lipopolysaccharide on neurogenesis in the hippocampus of rats.

Neurogenesis has been generally accepted to happen in the subventricular zone lining the lateral ventricular and subgranular zone (SGZ) in the hippocampus of adult mammalian brain. Recent studies have reported that inflammatory stimuli, such as injection of lipopolysaccharide (LPS), impair neurogenesis in the SGZ. Taurine, a sulfur-containing b-amino acid, is a major free intracellular amino acid in many tissues of mammals and having various supplementary effects on the mammalian body functions including the brain. Recently, it has been also reported that taurine levels in the brain significantly increase under stressful conditions. The present study was aimed to evaluate the possible beneficial effects of taurine on the neurogenesis in the SGZ under the condition of acute inflammatory stimuli by LPS. Adult male rats were intraperitoneally injected with taurine once a day for 39 days. Twenty-four hours before the animals were sacrificed on the last day of taurine treatment, LPS was injected simultaneously with bromodeoxyuridine (BrdU). Immunohistochemistry for BrdU, Ki67, and Iba-1 in the brain was performed, and serum levels of TNF-a and IL-1b 2 h after LPS injection were determined. The results showed that LPS significantly decreased the number of immunoreactive cells for both BrdU and Ki67 in the SGZ, while increased that for Iba-1, all of which were restored by taurine administration. Meanwhile, the serum concentrations of TNF-a and IL-1b were significantly increased, which were significantly attenuated by taurine administration. These results suggest that taurine effectively maintains neurogenesis in the SGZ under the acute infectious condition by attenuating the increase of microgliosis in the hippocampus as well as proinflammatory cytokines in the peripheral circulation.

Adv Exp Med Biol. 2013;775:111-9

Taurine resumed neuronal differentiation in arsenite-treated N2a cells through reducing oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction.

The goal of the study is to investigate the preventive effect of taurine against arsenite-induced arrest of neuronal differentiation in N2a cells. Our results revealed that taurine reinstated the neurite outgrowth in arsenite-treated N2a cells. Meanwhile, arsenite-induced oxidative stress and mitochondrial dysfunction as well as degradation of mitochondria DNA (mtDNA) were also inhibited by co-treatment of taurine. Since oxidative stress and mitochondrial dysfunction is closely associated with endoplasmic reticulum (ER) stress, we further examined indicators of ER stress, 78 kDa glucose-regulated protein (GRP78), and C/EBP-homologous protein (CHOP) protein expression. The results demonstrated that taurine significantly reduced arsenite-induced ER stress in N2a cells. In the parallel experiment, arsenite-induced disruption of intracellular calcium homeostasis was also ameliorated by taurine. The proven bio-function of taurine preserved a preventive effect against deleteriously cross-talking between oxidative stress, mitochondria, and ER. Overall, the results of the study suggested that taurine reinstated neuronal differentiation by inhibiting oxidative stress, ER stress, and mitochondrial dysfunction in arsenite-treated N2a cells.

Amino Acids. 2015 Apr;47(4):735-44

Endothelial Dysfunction

Serum amyloid A induces endothelial dysfunction in porcine coronary arteries and human coronary artery endothelial cells.

The objective of this study was to determine the effects and mechanisms of serum amyloid A (SAA) on coronary endothelial function. Porcine coronary arteries and human coronary arterial endothelial cells (HCAECs) were treated with SAA (0, 1, 10, or 25 microg/ml). Vasomotor reactivity was studied using a myograph tension system. SAA significantly reduced endothelium-dependent vasorelaxation of porcine coronary arteries in response to bradykinin in a concentration-dependent manner. SAA significantly decreased endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein levels as well as NO bioavailability, whereas it increased ROS in both artery rings and HCAECs. In addition, the activities of internal antioxidant enzymes catalase and SOD were decreased in SAA-treated HCAECs. Bio-plex immunoassay analysis showed the activation of JNK, ERK2, and IkappaB-alpha after SAA treatment. Consequently, the antioxidants seleno-l-methionine and Mn(III) tetrakis-(4-benzoic acid)porphyrin and specific inhibitors for JNK and ERK1/2 effectively blocked the SAA-induced eNOS mRNA decrease and SAA-induced decrease in endothelium-dependent vasorelaxation in porcine coronary arteries. Thus, SAA at clinically relevant concentrations causes endothelial dysfunction in both porcine coronary arteries and HCAECs through molecular mechanisms involving eNOS downregulation, oxidative stress, and activation of JNK and ERK1/2 as well as NF-kappaB. These findings suggest that SAA may contribute to the progress of coronary artery disease.

Am J Physiol Heart Circ Physiol. 2008 Dec;295(6):H2399-408

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein.

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25µg/ml) decreased nitric oxide ((•)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10µg/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(•-)) production. Gene expression for arginase-1, nuclear factor kB (NF-kB), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (•)NO bioavailability by stimulating O(2)(•-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, µg/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(•-) production; decreased TF, NF-kB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (•)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.

Free Radic Biol Med. 2011 Oct 1;51(7):1390-8

Effects of hesperidin on cyclic strain-induced endothelin-1 release in human umbilical vein endothelial cells.

1. Hesperidin, a member of the flavanone group of flavonoids, can be isolated in large amounts from the rinds of some citrus species and has been reported to have antihypotensive and vasodilator properties. However, the mechanism of action of hesperidin in the prevention and treatment of vascular diseases remains unclear. 2. The vascular endothelium can produce potent contracting factors, such as endothelin (ET)-1, and endothelium-derived relaxing factors, such as nitric oxide (NO). The aims of the present study were to test the hypothesis that hesperidin may alter strain-induced ET-1 secretion and NO production and to identify the putative underlying signalling pathways in human umbilical vein endothelial cells (HUVEC). 3. Hesperidin (10 and 100 micromol/L) inhibited strain-induced ET-1 secretion. Hesperidin also inhibited strain-induced increases in the formation of reactive oxygen species and extracellular signal-regulated kinase (ERK) phosphorylation. 4. Hesperidin treatment of HUVEC enhanced NO production, endothelial NO synthase (eNOS) activity and the phosphorylation of eNOS and Akt. Furthermore, hesperidin modulated strain-induced ET-1 release and suppressed ERK phosphorylation in part via the NO/protein kinase G pathway. 5. In summary, we have demonstrated that hesperidin inhibits strain-induced ET-1 secretion and enhances NO production in HUVEC.

Clin Exp Pharmacol Physiol. 2008 Aug;35(8):938-43

Distinct effects of naringenin and hesperetin on nitric oxide production from endothelial cells.

Diets rich in citrus and citrus-based products have been negatively correlated with the risk of cardiovascular disease, but so far no studies have been conducted to determine whether naringenin and hesperetin, two major flavanones in citrus plants, influence endothelium nitric oxide (NO) production. The aim of this study is to clarify estrogenic activities of naringenin and hesperetin and to examine whether they affect endothelial NO production via estrogen receptor (ER) activation. Both naringenin and hesperetin were observed to promote growth of MCF-7 cells under greatly reduced estrogen conditions and to suppress estrogen-induced response. Naringenin activated both ERalpha and ERbeta, whereas hesperetin exhibited stronger potential to activate ERalpha rather than ERbeta. Hesperetin, but not naringenin, increased NO releases from human umbilical vein endothelial cells in a dose-dependent manner. Hesperetin-induce responses were suppressed by ICI 182,780 and actinomycin D. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western-blotting analysis revealed that hesperetin up-regulated endothelium nitric oxide synthase (eNOS) expression. These results suggested that hesperetin exerts an antiatherogenic effect, in part, via ER-mediated eNOS expression and subsequent increase of endothelial NO production. Distinct effects of naringenin and hesperetin on NO production also imply that ERalpha might play the major role in estrogen-induced eNOS expression. However, the inefficacy of naringenin on NO production remains to be elaborately studied. Our findings add more proof to the molecular explanations for the health benefits of citrus used to prevent cardiovascular disease, especially for postmenopausal women.

J Agric Food Chem . 2008 Feb 13;56(3):824-9

Hesperidin supplementation modulates inflammatory responses following myocardial infarction.

OBJECTIVE: A growing number of studies have suggested a crucial role for a variety of inflammatory mediators in myocardial infarction. Recently, several flavonoids have been shown to have cardioprotective and anti-inflammatory properties. Therefore, the aim of this study was to investigate the effect of hesperidin-a common constituent of citrus fruits-on the serum levels of inflammatory markers and adipocytocines in patients with myocardial infarction. METHODS: Seventy-five patients with myocardial infarction were participated in this randomized, double-blind controlled clinical trial and were assigned to 2 intervention and control groups. Subjects consumed 600 mg/d pure hesperidin supplement and placebo in the intervention and control groups, respectively, for 4 weeks. Serum concentrations of inflammatory markers and adipocytocines were measured at baseline and at the end of the intervention. RESULTS: Consumption of 600 mg/day hesperidin significantly decreased the serum levels of E-selectin and increased adiponectin and high-density lipoprotein cholesterol (HDL-C) concentrations in patients with myocardial infarction. The improvement in other inflammatory markers, such as interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), leptin, and other lipid profile was also observed at the end of the intervention, compared to the baseline values, but the difference between the hesperidin and placebo groups was not statistically significant (p > 0.05). CONCLUSION: Hesperidin supplementation could compensate for decreased levels of adiponectin and HDL-C and increased levels of E-selectin in patients with myocardial infarction. These results support the concept that certain flavonoids in the diet can be associated with significant health benefits, including heart health.

J Am Coll Nutr. 2015 May-Jun;34(3):205-11

Citrus polyphenol hesperidin stimulates production of nitric oxide in endothelial cells while improving endothelial function and reducing inflammatory markers in patients with metabolic syndrome.

CONTEXT: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. OBJECTIVE: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. DESIGN, SETTING, AND INTERVENTIONS: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). MAIN OUTCOME MEASURE: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. RESULTS: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H(2)O(2). Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-a treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). CONCLUSIONS: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

J Clin Endocrinol Metab . 2011 May;96(5):E782-92

Short-term effects of glucosyl hesperidin and hesperetin on blood pressure and vascular endothelial function in spontaneously hypertensive rats.

Glucosyl hesperidin (G-hesper-idin) is a water-soluble derivative of hesperidin. In the present study, the short-term effects of G-hesperidin and hesperetin, a putative metabolite of G-hesperidin, in spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto rats (WKY) were investigated. Single oral administration of G-hesperidin (10 to 50 mg/kg) induced a dose-dependent reduction in systolic blood pressure (SBP) in SHR, but had no effects in WKY. Intraperitoneal injection of hesperetin (50 mg/kg) into SHR also caused a significant reduction in SBP. The depressor effect was significantly inhibited by a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester. Moreover, hesperetin (10(-5) M) enhanced endothelium-dependent relaxation induced by acetylcholine, but had no effect on endothelium-independent relaxation induced by sodium nitroprusside in isolated aortas from SHR. These data suggest that the hypotensive effect of hesperetin in SHR is associated with nitric oxide-mediated vasodilation. Therefore, this effect may be involved in the mechanisms by which G-hesperidin lowers blood pressure in hypertension.

J Nutr Sci Vitaminol (Tokyo) . 2008 Feb;54(1):95-8

Hesperetin inhibits rat coronary constriction by inhibiting Ca(2+) influx and enhancing voltage-gated K(+) channel currents of the myocytes.

Hesperetin (HSP, one of the most common flavonoids in Citrus) has been reported to possess many benificial effects and is indicated for many diseases both as a therapeutic drug and as a supplement. Although its vascular effects have been extensively studied, little is known about its effects and the underlying mechanisms on coronary artery. In the present study, the myogenic effects of HSP were studied with a wire myograph in isolated rat coronary artery (RCA). Molecular probe and the patch clamp technique were used to study effects of HSP on intracellular free Ca(2+) concentration, inward Ca(2+) currents through L-type voltage-gated Ca(2+) channels (LVGC) and outward K(+) currents through voltage-gated K(+) channels (KV). HSP (0.01-0.1mM) concentration-dependently depressed concentration-contraction curves of both KCl and thromboxane receptor agonist 9,11-Dideoxy-9a,11a-methanoepoxy prostaglandin F2a (U46619), and relaxed RCA precontracted by the both vasoconstrictors. The vasospasmolytic effect was more potent in KCl- than in U46619-induced contraction. The vasorelaxation was attenuated by 4-aminopyridine, a specific KV inhibitor, but not affected by NG-nitro-L-arginine methylester ester, indomethacin, glibenclamide, iberiotoxin, BaCl2 or endothelium denudation. At the same concentrations, HSP inhibited extracellular Ca(2+) influx-induced contraction, reduced intracellular free Ca(2+) concentration, inhibited inward Ca(2+) currents through LVGC and increased outward K(+) currents through KV in the vascular smooth muscle cells (VSMCs) freshly isolated from RCA. Collectively, our results show that HSP is vasospasmolytic in RCA and suggest that the vasospasmolysis is mediated by inhibition of LVGC and enhancement of KV currents in RCA VSMCs.

Eur J Pharmacol . 2014 Jul 15;735:193-201

Endothelial function predicts progression of carotid intima-media thickness.

BACKGROUND: Endothelial dysfunction develops early and has been shown to predict the development of clinical complications of atherosclerosis. However, the relationship between early endothelial dysfunction and the progression of arterial disease in the general population is unknown. We investigated endothelial dysfunction, risk factors, and progression of carotid intima-media thickness (cIMT) in late-middle-aged individuals at low to intermediate cardiovascular risk in a prospective study between 1997 and 2005. METHODS AND RESULTS: Brachial artery flow-mediated dilatation and cIMT were measured in 213 nonsmoking British civil servants recruited from a prospective cohort (Whitehall II study). Participants (age, 45 to 66 years) were free of clinical cardiovascular disease and diabetes mellitus. Risk factors and Framingham Risk Score were determined at baseline. cIMT was repeated 6.2+/-0.4 years later. At baseline, age, blood pressure, low-density lipoprotein cholesterol, and Framingham Risk Score correlated with cIMT. However, only flow-mediated dilatation, not risk factors or Framingham Risk Score, was associated with average annual progression of cIMT. This relationship remained significant after adjustment for risk factors whether entered as separate variables or as Framingham Risk Score. Further adjustment for waist circumference, triglycerides, and employment grade had no significant effect. CONCLUSIONS: Systemic endothelial function was associated with progression of preclinical carotid arterial disease over a 6-year period and was more closely related to cIMT changes than conventional risk factors. Thus, the relationship between endothelial dysfunction and adverse outcome is likely to be due not only to destabilization of established disease in high-risk populations but also to its impact on the evolution of the atherosclerotic substrate. Flow-mediated dilatation testing provides an integrated vascular measure that may aid the prediction of structural disease evolution and represents a potential short- to intermediate-term outcome measure for evaluation of preventive treatment strategies.

Circulation . 2009 Feb 24;119(7):1005-12

Monocytes induce E-selectin gene expression in endothelial cells: role of CD11/CD18 and extracellular matrix proteins.

E-selectin is an endothelium-specific inducible adhesion molecule which binds several inflammatory cell types, including neutrophils, monocytes, natural killer cells and a subset of memory T cells. E-selectin is important in the initial rolling interaction of these cells on inflamed endothelium. The transient kinetics of E-selectin induction in vitro contrast with in vivo observations of prolonged expression of this adhesion molecule in chronic inflammation. This raises the possibility that signals generated within inflammatory tissues are more complex than the agonists used to activate endothelial cells in vitro. We investigated whether adhesive interactions with extravasating monocytes are able to provide activating signals that can induce E-selectin expression on endothelium, and prolong the response to cytokine stimulation. We report that co-culture with monocytes led to transcriptional activation of the E-selectin gene in endothelial cells, and marked enhancement of the response to substimulatory concentrations of interleukin-1. In addition, the presence of monocytes resulted in prolonged up-regulation of E-selectin. Induction of E-selectin by monocytes was inhibited when cell contact between monocytes and endothelium was prevented (80 +/- 8% inhibition, p < 0.001, n = 4). Monoclonal antibody (mAb) against tumor necrosis factor (TNF) was able to abolish 57.2 +/- 9.7% of the response (p < 0.01, n = 4). The ability of adherent monocytes to induce sustained E-selectin expression in endothelial cells could not be reproduced either by supernatants harvested from monocytes cultured for 18 h, or by maximal concentrations of TNF. The induction of E-selectin in monocyte/endothelium co-cultures was inhibited by mAb to CD11b, but not by those directed against VLA-4 or L-selectin. Extracellular matrix molecules may also play a role in adhesion-dependent cellular activation, as inclusion of soluble collagen type I led to significant reduction in E-selectin expression in monocyte/endothelium co-cultures. We conclude that adhesive interactions between monocytes and endothelial cells provide a source of signals which influence the activation state of the endothelium, and consequently, the continued influx of inflammatory cells.

Eur J Immunol. 1996 Dec;26(12):2944-51

Fucoidan

Anti-influenza A virus characteristics of a fucoidan from sporophyll of Undaria pinnatifida in mice with normal and
compromised immunity.

Undaria pinnatifida, an edible brown alga, contains fucoidan (FuC), a sulfated polysaccharide, that inhibited the in vitro replication of influenza A virus, and stimulated both innate and adaptive immune defense functions in virus-infected mice. In the present study, the effects of oral administration of FuC were evaluated on influenza virus infection in immunocompetent and immunocompromised mice, where the efficacy of FuC was demonstrated in reducing viral replication, decreasing weight loss and mortality, and prolonging survival. Oral FuC resulted in increased neutralizing antibody production in the mucosa and blood. In contrast, while suppressing virus yields in mice more markedly than FuC, oseltamivir significantly reduced the neutralizing antibody titers in both the mucosa and blood. In immunocompromised mice, drug-resistant viruses frequently recovered after oseltamivir treatment; no resistant viruses were isolated from FuC-treated mice. FuC could be a candidate for the development of new therapeutic options including its combination with neuraminidase inhibitors such as oseltamivir.

Microbes Infect. 2013 Apr;15(4):302-9

Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida.

BACKGROUND: We showed that fucoidan, extracted from dietary seaweed, could inhibit tumor growth. However, the mechanism of Mekabu (Sporophyll of Undaria pinnatifida) fucoidan antitumor activity and how it enhances the immune response remains unknown. MATERIALS AND METHODS: We examined the effect of Mekabu fucoidan in P-388 tumor-bearing mice and in T cell-mediated NK cell activity in normal mice. RESULTS: The survival of mice was prolonged when Mekabu fucoidan was administered for 4 days before tumor cell inoculation, compared with non-treated mice. Fucoidan significantly enhanced the cytolytic activity of NK cells and increased the amount of IFN-gamma produced by T cells up to about 2-fold compared with non-treated mice. CONCLUSION: The anti-tumor effect of Mekabu fucoidan appears to be mediated by IFN-gamma-activated NK cells.

In Vivo. 2003 May-Jun;17(3):245-9

Anti-metastasis effect of fucoidan from Undaria pinnatifida sporophylls in mouse hepatocarcinoma Hca-F cells.

Metastasis is one of the major causes of cancer-related death. It is a complex biological process involving multiple genes, steps, and phases. It is also closely connected to many biological activities of cancer cells, such as growth, invasion, adhesion, hematogenous metastasis, and lymphatic metastasis. Fucoidan derived from Undaria pinnatifida sporophylls (Ups-fucoidan) is a sulfated polysaccharide with more biological activities than other fucoidans. However, there is no information on the effects of Ups-fucoidan on tumor invasion and metastasis. We used the mouse hepatocarcinoma Hca-F cell line, which has high invasive and lymphatic metastasis potential in vitro and in vivo, to examine the effect of Ups-fucoidan on cancer cell invasion and metastasis. Ups-fucoidan exerted a concentration- and time-dependent inhibitory effect on tumor metastasis in vivo and inhibited Hca-F cell growth, migration, invasion, and adhesion capabilities in vitro. Ups-fucoidan inhibited growth and metastasis by downregulating vascular endothelial growth factor (VEGF) C/VEGF receptor 3, hepatocyte growth factor/c-MET, cyclin D1, cyclin-dependent kinase 4, phosphorylated (p) phosphoinositide 3-kinase, p-Akt, p-extracellular signal regulated kinase (ERK) 1/2, and nuclear transcription factor-kB (NF-kB), and suppressed adhesion and invasion by downregulating L-Selectin, and upregulating protein levels of tissue inhibitor of metalloproteinases (TIMPs). The results suggest that Ups-fucoidan suppresses Hca-F cell growth, adhesion, invasion, and metastasis capabilities and that these functions are mediated through the mechanism involving inactivation of the NF-kB pathway mediated by PI3K/Akt and ERK signaling pathways.

PLoS One . 2014 Aug 27;9(8):e106071

Fucoidan from Macrocystis pyrifera has powerful immune-modulatory effects compared to three other fucoidans.

Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, such as promoting activation of dendritic cells (DCs), natural killer (NK) cells and T cells, and enhancing anti-viral and anti-tumor responses. However, the immune-modulatory effect of fucoidan from different seaweed extracts has not been thoroughly analyzed and compared. We analyzed fucoidans obtained from Ascophyllum nodosum (A. nodosum), Macrocystis pyrifera (M. pyrifera), Undaria pinnatifida (U. pinnatifida) and Fucus vesiculosus (F. vesiculosus) for their effect on the apoptosis of human neutrophils, activation of mouse NK cells, maturation of spleen DCs, proliferation and activation of T cells, and the adjuvant effect in vivo. Fucoidans from M. pyrifera and U. pinnatifida strongly delayed human neutrophil apoptosis at low concentration, whereas fucoidans from A. nodosum and F. vesiculosus delayed human neutrophil apoptosis at higher concentration. Moreover, fucoidan from M. pyrifera promoted NK cell activation and cytotoxic activity against YAC-1 cells. In addition, M. pyrifera fucoidan induced the strongest activation of spleen DCs and T cells and ovalbumin (OVA) specific immune responses compared to other fucoidans. These data suggest that fucoidan from M. pyrifera can be potentially useful as a therapeutic agent for infectious diseases, cancer and an effective adjuvant for vaccine.

Mar Drugs. 2015 Feb 19;13(3):1084-104

Fucoidan can function as an adjuvant in vivo to enhance dendritic cell maturation and function and promote antigen-specific T cell immune responses.

Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, including promoting antigen uptake and enhancing anti-viral and anti-tumor effects. However, the effect of fucoidan in vivo, especially its adjuvant effect on in vivo anti-tumor immune responses, was not fully investigated. In this study, we investigated the effect of fucoidan on the function of spleen dendritic cells (DCs) and its adjuvant effect in vivo. Systemic administration of fucoidan induced up-regulation of CD40, CD80 and CD86 expression and production of IL-6, IL-12 and TNF-a in spleen cDCs. Fucoidan also promoted the generation of IFN-g-producing Th1 and Tc1 cells in an IL-12-dependent manner. When used as an adjuvant in vivo with ovalbumin (OVA) antigen, fucoidan promoted OVA-specific antibody production and primed IFN-g production in OVA-specific T cells. Moreover, fucoidan enhanced OVA-induced up-regulation of MHC class I and II on spleen cDCs and strongly prompted the proliferation of OVA-specific CD4 and CD8 T cells. Finally, OVA immunization with fucoidan as adjuvant protected mice from the challenge with B16-OVA tumor cells. Taken together, these results suggest that fucoidan can function as an adjuvant to induce Th1 immune response and CTL activation, which may be useful in tumor vaccine development.

PLoS One. 2014 Jun 9;9(6):e99396

Suppression of Th2 immune responses by mekabu fucoidan from Undaria pinnatifida sporophylls.

BACKGROUND: We demonstrated that mekabu fucoidan obtained from Undaria pinnatifida (Up) sporophylls augments the type 1 T-helper (Th1) cell response in normal BALB/c mice. In this study, we examined the effects of the fucoidan of mekabu on the type 2 T-helper (Th2) response in bronchoalveolar lavage fluid (BALF) after ovalbumin (OVA) aerosol challenge. METHODS: Mekabu fucoidan (50 mg/kg) was injected intraperitoneally into BALB/c mice for 4 days, and then the mice were sensitized with 50 microg/mouse of OVA plus alum (1 mg/mouse) 1 and 8 days later. The mice were challenged with OVA delivered using a nebulizer 7, 8 and 9 days after the second challenge with OVA plus alum. After 24 h, we assessed T cell responses in BALF by measuring the amount of Th2 cytokines (IL-4, IL-5, IL-13) and gamma-interferon (IFN-gamma) produced by Th1 cells. RESULTS: The production of Th2 cytokines was suppressed (p < 0.05), and the amount of IFN-gamma was not increased in the mice treated with mekabu fucoidan. Anti-OVA immunoglobulin E (IgE) and IgE levels in serum determined after challenge with aerosolized OVA at the end of the experiment were lower (p < 0.05) in the treated than in the control mice. CONCLUSIONS: The pulmonary inflammation was relieved by mekabu fucoidan, which also downregulated Th2-dominated responses. These results indicate that mekabu fucoidan modulates Th2 responses and might be useful for treating allergic inflammation.

Int Arch Allergy Immunol . 2005 Aug;137(4):289-94

Therapies from fucoidan; multifunctional marine polymers.

Published research on fucoidans increased three fold between 2000 and 2010. These algal derived marine carbohydrate polymers present numerous valuable bioactivities. This review discusses the role for fucoidan in the control of acute and chronic inflammation via selectin blockade, enzyme inhibition and inhibiting the complement cascade. The recent data on toxicology and uptake of fucoidan is detailed together with a discussion on the comparative activities of fractions of fucoidan from different sources. Recent in vivo, in vitro and clinical research related to diverse clinical needs is discussed. Targets include osteoarthritis, kidney and liver disease, neglected infectious diseases, hemopoietic stem cell modulation, protection from radiation damage and treatments for snake envenomation. In recent years, the production of well characterized reproducible fucoidan fractions on a commercial scale has become possible making therapies from fucoidan a realizable goal.

Mar Drugs . 2011;9(10):1731-60.

Fucoidan and cancer: a multifunctional molecule with anti-tumor potential.

There is a wide variety of cancer types yet, all share some common cellular and molecular behaviors. Most of the chemotherapeutic agents used in cancer treatment are designed to target common deregulated mechanisms within cancer cells. Many healthy tissues are also affected by the cytotoxic effects of these chemical agents. Fucoidan, a natural component of brown seaweed, has anti-cancer activity against various cancer types by targeting key apoptotic molecules. It also has beneficial effects as it can protect against toxicity associated with chemotherapeutic agents and radiation. Thus the synergistic effect of fucoidan with current anti-cancer agents is of considerable interest. This review discusses the mechanisms by which fucoidan retards tumor development, eradicates tumor cells and synergizes with anti-cancer chemotherapeutic agents. Challenges to the development of fucoidan as an anti-cancer agent will also be discussed.

Mar Drugs. 2015 Apr 14;13(4):2327-46

Sulfated polysaccharide fucoidan ameliorates experimental autoimmune myocarditis in rats.

Homing of cardiac myosin-specific CD4-positive T cells into the myocardium is the initial pathologic event of experimental autoimmune myocarditis (EAM). Subsequently, various bystander inflammatory cells are recruited into the myocardium crossing vascular endothelial cell walls. Sulfated polysaccharide fucoidan binds selectin nonselectively and blocks its function. Therefore, this study was designed to evaluate whether in vivo fucoidan treatment can improve EAM. A 21-day infusion of physiological saline or fucoidan was administrated intraperitoneally to the rats with sham operation (sham-saline, n = 5; sham-fucoidan, n = 6) or those with cardiac myosin injection (EAM-saline, n = 10; EAM-fucoidan, n = 10). After 3 weeks, fucoidan treatment improved left ventricular ejection fraction (79.04 ± 2.81 vs 65.94% ± 3.22%; P < .01 vs EAM-saline) with a reduced ratio of heart weight to body weight (4.016 ± 0.239 vs 4.975 ± 0.252 mg/g; P < .05 vs EAM-saline) in EAM. Furthermore, fucoidan treatment decreased serum levels of BNP (292.0 ± 53.4 vs 507.4 ± 89.2 ng/mL; P < .05 vs EAM-saline) and the myocarditis area (31.66 ± 1.53 vs 42.51% ± 3.24%; P < .01 vs EAM-saline) in EAM. These beneficial effects of fucoidan were accompanied by inhibition of both macrophage and CD4-positive T-cell infiltration into the myocardium. Fucoidan, a nonselective selectin blocker, attenuates the progression of EAM. This observation may be explained, at least in part, by blocking the extravasation of inflammatory cells into the myocardium.

J Cardiovasc Pharmacol Ther . 2011 Mar;16(1):79-86

A combined phase I and II open label study on the effects of a seaweed extract nutrient complex on osteoarthritis.

BACKGROUND: Isolated fucoidans from brown marine algae have been shown to have a range of anti-inflammatory effects. PURPOSE: This present study tested a Maritech((R)) extract formulation, containing a blend of extracts from three different species of brown algae, plus nutrients in an open label combined phase I and II pilot scale study to determine both acute safety and efficacy in osteoarthritis of the knee. PATIENTS AND METHODS: Participants (n = 12, five females [mean age, 62 +/- 11.06 years] and seven males [mean age, 57.14 +/- 9.20 years]) with a confirmed diagnosis of osteoarthritis of the knee were randomized to either 100 mg (n = 5) or 1000 mg (n = 7) of a Maritech((R)) extract formulation per day. The formulation contained Maritech((R)) seaweed extract containing Fucus vesiculosis (85% w/w), Macrocystis pyrifera (10% w/w) and Laminaria japonica (5% w/w) plus vitamin B6, zinc and manganese. Primary outcome was the average comprehensive arthritis test (COAT) score which is comprised of four sub-scales: pain, stiffness, difficulty with physical activity and overall symptom severity measured weekly. Safety measures included full blood count, serum lipids, liver function tests, urea, creatinine and electrolytes determined at baseline and week 12. All adverse events were recorded. RESULTS: Eleven participants completed 12 weeks and one completed 10 weeks of the study. Using a multilevel linear model, the average COAT score was reduced by 18% for the 100 mg treatment and 52% for the 1,000 mg dose at the end of the study. There was a clear dose response effect seen between the two treatments (P </= 0.0005) on the average COAT score and each of the four COAT subscales (pain, stiffness, difficulty with physical activity and overall symptom severity) (P </= 0.05). The preparation was well tolerated and the few adverse events were unlikely to be related to the study medication. There were no changes in blood parameters measured over the course of the study with the exception of an increase in serum albumin which was not clinically significant. CONCLUSION: The seaweed extract nutrient complex when taken orally over twelve weeks decreased the symptoms of osteoarthritis in a dose-dependent manner. It was demonstrated to be safe to use over the study period at the doses tested. The efficacy of the preparation now needs to be demonstrated in a phase III randomized controlled trial (RCT).

Biologics. 2010 Mar 24;4:33-44

Thymus Regeneration

Involution patterns of the human thymus. Size of the cortical area as a function of age.

Thymic tissue obtained at autopsy from 123 victims of sudden death, ranging from neonates to individuals older than 80 years and predominantly male, was studied with histometric techniques, i.e. a combination of test point analysis and planimetry on unit optical fields. The pattern of ‘natural’ age-dependent involution of the thymic cortex was examined using computerized mathematical models. The range of variations of results was greatest in children and young adults, followed by the very old, then the middle age group. In a first approximation, regression for thymic cortical volume in individuals older than 15 years corresponded better to a negative exponential than to a negative linear function of age. Best fits for the data suggest at least a two-component negative exponential function of age, with a steeper slope of the regression for individuals beyond the age of 30 years. Extrapolation on a log-normal plot of regression lines for thymic cortical involution points to near-zero values at an age range below the estimated maximum human life span, corresponding to the steepest slope of survival curves in Western Europe.

Clin Exp Immunol . 1982 Feb;47(2):497-504

Tissue engineering and regenerative medicine: past, present, and future.

Tissue and organ repair still represents a clinical challenge. Tissue engineering and regenerative medicine (TERM) is an emerging field focused on the development of alternative therapies for tissue/organ repair. This highly multidisciplinary field, in which bioengineering and medicine merge, is based on integrative approaches using scaffolds, cell populations from different sources, growth factors, nanomedicine, gene therapy, and other techniques to overcome the limitations that currently exist in the clinics. Indeed, its overall objective is to induce the formation of new functional tissues, rather than just implanting spare parts. This chapter aims at introducing the reader to the concepts and techniques of TERM. It begins by explaining how TERM have evolved and merged into TERM, followed by a short overview of some of its key aspects such as the combinations of scaffolds with cells and nanomedicine, scaffold processing, and new paradigms of the use of stem cells for tissue repair/regeneration, which ultimately could represent the future of new therapeutic approaches specifically aimed at clinical applications.

Int Rev Neurobiol. 2013;108:1-33

An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts.

A central goal of regenerative medicine is to generate transplantable organs from cells derived or expanded in vitro. Although numerous studies have demonstrated the production of defined cell types in vitro, the creation of a fully intact organ has not been reported. The transcription factor forkhead box N1 (FOXN1) is critically required for development of thymic epithelial cells (TECs), a key cell type of the thymic stroma. Here, we show that enforced Foxn1 expression is sufficient to reprogramme fibroblasts into functional TECs, an unrelated cell type across a germ-layer boundary. These FOXN1-induced TECs (iTECs) supported efficient development of both CD4(+) and CD8(+) T cells in vitro. On transplantation, iTECs established a complete, fully organized and functional thymus, that contained all of the TEC subtypes required to support T-cell differentiation and populated the recipient immune system with T cells. iTECs thus demonstrate that cellular reprogramming approaches can be used to generate an entire organ, and open the possibility of widespread use of thymus transplantation to boost immune function in patients.

Nat Cell Biol. 2014 Sep;16(9):902-8

Regenerative medicine strategies.

Applications of regenerative medicine technology may offer novel therapies for patients with injuries, end-stage organ failure, or other clinical problems. Currently, patients suffering from diseased and injured organs can be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly as the population ages and new cases of organ failure increase. Scientists in the field of regenerative medicine and tissue engineering are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The stem cell field is also advancing rapidly, opening new avenues for this type of therapy. For example, therapeutic cloning and cellular reprogramming may one day provide a potentially limitless source of cells for tissue engineering applications. While stem cells are still in the research phase, some therapies arising from tissue engineering endeavors have already entered the clinical setting successfully, indicating the promise regenerative medicine holds for the future.

J Pediatr Surg . 2012 Jan;47(1):17-28

A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (~92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.

Tissue Eng Part A . 2012 Dec;18(23-24):2437-52

Perspectives on whole-organ assembly: moving toward transplantation on demand.

There is an ever-growing demand for transplantable organs to replace acute and chronically damaged tissues. This demand cannot be met by the currently available donor organs. Efforts to provide an alternative source have led to the development of organ engineering, a discipline that combines cell biology, tissue engineering, and cell/organ transplantation. Over the last several years, engineered organs have been implanted into rodent recipients and have shown modest function. In this article, we summarize the most recent advances in this field and provide a perspective on the challenges of translating this promising new technology into a proven regenerative therapy.

J Clin Invest . 2012 Nov;122(11):3817-23

Abdominal organ bioengineering: current status and future perspectives.

Organ transplantation represents one of the major milestones of modern medicine and surgical practice in terms of life-years prolonged and quality of life offered for chronic patients. Each year over 100,000 donor organ transplants are performed worldwide. In spite of the rapid advancement and expansion of this niche, it has become a victim of its own success as the donor supply is far oustripped by the demand for replacement organs. Furthermore, current methods only allow for successful transplantation in the setting of life-long, aggressive immunosuppression protocols which enhances the incidence of secondary neoplasm and other associated sequelae. Against this background, recent advances in the fields of regenerative medicine, tissue engineering, and cellular biology have coalesced into a promising new avenue of investigation involving the fabrication of de novo, transplantable organs using autologous cells. Donor organs are stripped of their native cellular material leaving only acellular, extracellular matrix constructs behind. These constructs can then be recellularized with a patient’s own cells in order to form transplantable organs that do not require immunosuppression. Furthermore, in theory, these methods could provide a potentially inexhaustible source of organs to meet the growing need for viable transplants. In this review, we describe these methods as well as contemporary successes for various organ systems.

Minerva Chir. 2015 Feb;70(1):43-55

Generation of human-induced pluripotent stem cells.

Pluripotent cells, such as embryonic stem cells, are invaluable tools for research and can potentially serve as a source of cell- and tissue-replacement therapy. Rejection after transplantation of cells and tissue derived from embryonic stem cells is a significant obstacle to their clinical use. Recently, human somatic cells have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Human iPS cells are a potential source of patient-specific pluripotent stem cells that would bypass immune rejection. iPS cells can also be used to study diseases for which there are no adequate human in vitro or animal models. In this protocol, we describe how to establish primary human fibroblasts lines and how to derive iPS cells by retroviral transduction of reprogramming factors. Overall, it takes 2 months to complete reprogramming human primary fibroblasts starting from biopsy.

Nat Protoc. 2008;3(7):1180-6

Decellularization for whole organ bioengineering.

Organ transplantation in an orthotopic location is the current treatment for end-stage organ failure. However, the need for transplantable organs far exceeds the number of available donor organs. As a result, new options, such as tissue engineering and regenerative medicine, have been explored to achieve functional organ replacement. Although there have been many advances in the laboratory leading to the reconstruction of tissue and organ structures in vitro, these efforts have fallen short of producing organs that contain intact vascular networks capable of nutrient and gas exchange and are suitable for transplantation. Recently, advances in whole organ decellularization techniques have enabled the fabrication of scaffolds for engineering new organs. These scaffolds, consisting of naturally-derived extracellular matrix (ECM), provide biological signals and maintain tissue microarchitecture, including intact vascular systems that could integrate into the recipient’s circulatory system. The decellularization techniques have led to the development of scaffolds for multiple organs, including the heart, liver, lung and kidney. While the experimental studies involving the use of decellularized organ scaffolds are encouraging, the translation of whole organ engineering into the clinic is still distant. This paper reviews recently described techniques used to decellularize whole organs such as the heart, lung, liver and kidney and describes possible methods for using these matrices for whole organ engineering.

Biomed Mater. 2013 Feb;8(1):014106

FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program.

T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs) drive T cell differentiation, education, and selection processes, while the thymocyte-dependent signals allow thymic epithelial cells (TECs) to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1, and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1) gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the post-natal thymus. An inborn null mutation in FOXN1 leads to Nude/severe combined immunodeficiency (SCID) phenotype in mouse, rat, and humans. In Foxn1 (-/-) nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.

Front Immunol . 2013 Jul 12;4:187

Renal bioengineering with scaffolds generated from human kidneys.

BACKGROUND: In 2012, about 16,487 people received kidney transplants in the USA whereas 95,022 candidates were on the waiting list at the end of the year. Moreover, more than 2,600 kidneys procured annually for transplantation are discarded for a variety of reasons. We hypothesize that this pool of discarded kidneys could in part meet the growing, urgent need for transplantable kidneys using current methods for organ bioengineering and regeneration and surgical transplantation. The recellularization of extracellular matrix (ECM) scaffolds has the potential to meet the uniquely ambitious engineering challenges posed by complex solid organs such as the kidney. SUMMARY: Attempts to manufacture and implant simpler, hollow structures such as bladders, vessels, urethras, and segments of the upper airways have been successful in the short and mid terms. However, the bioengineering of complex solid organs such as the kidney is a more challenging task that requires a different approach. In previous studies, we showed that decellularized porcine kidneys yield renal ECM scaffolds that preserve their basic architecture and structural components, support cell growth in vivo and in vitro, and maintain a patent vasculature capable of sustaining physiological blood pressure. In a subsequent report, using the same methods, we found that detergent-based decellularization of discarded human renal kidneys preserved their innate ECM framework, biochemical properties, and angiogenic capacity and - importantly - a patent vascular network. Furthermore, the process resulted in the clearance of immunogenic antigens, which has monumental implications for clinical outcomes in the long term in terms of graft rejection. Consequently, these kidneys show promise in bioengineering and transplantation. We refer to this avenue of research and development as ‘cell-scaffold technology’. KEY MESSAGES: In 2011, more than 4,700 patients died while on the waiting list for a kidney transplant. In this context, we believe that cell-scaffold technology has the potential to form a bridge between regenerative medicine and transplantation surgery. These methods, in theory, could provide a potentially inexhaustible source of transplantable organs. Unfortunately, current investigations are still in their very early stages and clinical translation is not immediately available in the short term. Thus, identifying the most important obstacles confronting cell-scaffold technology and focusing research efforts in this direction will be important for advancing the state of the art and meeting the clinical needs. We believe that cell-scaffold technology research and development would benefit greatly from a deeper understanding of the physiological mechanisms underlying the natural organogenesis, regeneration, and repair that characterize embryonic humans and simpler organisms. Furthermore, the importance of vascularization - the fundamental caveat of modern surgery - cannot be overstated, especially when discussing the implantation of de novo organs.

Nephron Exp Nephrol. 2014;126(2):119

Extracellular Matrix Scaffold Technology for Bioartificial Pancreas Engineering: State of the Art and Future Challenges.

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management.

J Diabetes Sci Technol . 2014 Jan 1;8(1):159-169

Regeneration of the aged thymus by a single transcription factor.

Thymic involution is central to the decline in immune system function that occurs with age. By regenerating the thymus, it may therefore be possible to improve the ability of the aged immune system to respond to novel antigens. Recently, diminished expression of the thymic epithelial cell (TEC)-specific transcription factor Forkhead box N1 (FOXN1) has been implicated as a component of the mechanism regulating age-related involution. The effects of upregulating FOXN1 function in the aged thymus are, however, unknown. Here, we show that forced, TEC-specific upregulation of FOXN1 in the fully involuted thymus of aged mice results in robust thymus regeneration characterized by increased thymopoiesis and increased naive T cell output. We demonstrate that the regenerated organ closely resembles the juvenile thymus in terms of architecture and gene expression profile, and further show that this FOXN1-mediated regeneration stems from an enlarged TEC compartment, rebuilt from progenitor TECs. Collectively, our data establish that upregulation of a single transcription factor can substantially reverse age-related thymic involution, identifying FOXN1 as a specific target for improving thymus function and, thus, immune competence in patients. More widely, they demonstrate that organ regeneration in an aged mammal can be directed by manipulation of a single transcription factor, providing a provocative paradigm that may be of broad impact for regenerative biology

Development. 2014 Apr;141(8):1627-37.

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