Peptide OM-LV20 promotes arteriogenesis induced by femoral artery ligature via the miR-29b-3p/VEGFA axis.

BACKGROUND AND AIMS: Therapeutic arteriogenesis is a promising direction for the treatment of ischemic disease caused by atherosclerosis. However, pharmacological or biological approaches to stimulate functional collateral vessels are not yet available. Identifying new drug targets to promote and explore the underlying mechanisms for therapeutic arteriogenesis is necessary. METHODS: Peptide OM-LV20 (20 ng/kg) was administered for 7 consecutive days on rat hindlimb ischemia model, collateral vessel growth was assessed by H&E staining, liquid latex perfusion, and specific immunofluorescence. In vitro, we detected the effect of OM-LV20 on human umbilical vein endothelial cells (HUVEC) proliferation and migration. After transfection, we performed quantitative real-time polymerase chain reaction, in situ-hybridization and dual luciferase reporters to assessed effective miRNAs and target genes. The proteins related to downstream signaling pathways were detected by Western blot. RESULTS: OM-LV20 significantly increased visible collateral vessels and endothelial nitric oxide synthase (eNOS), together with enhanced inflammation cytokine and monocytes/macrophage infiltration in collateral vessels. In vitro, we defined a novel microRNA (miR-29b-3p), and its inhibition enhanced proliferation and migration of HUVEC, as well as the expression of vascular endothelial growth factor A (VEGFA). OM-LV20 also promoted migration and proliferation of HUVEC, and VEGFA expression was mediated via inhibition of miR-29b-3p. Furthermore, OM-LV20 influenced the protein levels of VEGFR2 and phosphatidylinositol3-kinase (PI3K)/AKT and eNOS in vitro and invivo. CONCLUSIONS: Our data indicated that OM-LV20 enhanced arteriogenesis via the miR-29b-3p/VEGFA/VEGFR2-PI3K/AKT/eNOS axis, and highlighte the application potential of exogenous peptide molecular probes through miRNA, which could promote effective therapeutic arteriogenesis in ischemic conditions.

Betaine prevents cognitive dysfunction by suppressing hippocampal microglial activation in chronic social isolated male mice.

Chronic social isolation (SI) stress, which became more prevalent during the COVID-19 pandemic, contributes to abnormal behavior, including mood changes and cognitive impairment. Known as a functional nutrient, betaine has potent antioxidant and anti-inflammatory properties in vivo. However, whether betaine can alleviate the abnormal behavior induced by chronic SI in mice remains unknown. In this study, we investigated the efficacy of betaine in the treatment of behavioral changes and its underlying mechanism. Three-week-old male mice were randomly housed for 8 weeks in either group housing (GH) or SI. The animals were divided into normal saline-treated GH, normal saline-treated SI, and betaine-treated SI groups in the sixth week. The cognitive and depression-like behavior was determined in the eighth week. We found that long-term betaine administration improved cognitive behavior in SI mice but failed to prevent depression-like behavior. Moreover, long-term betaine administration inhibited hippocampal microglia over-activation and polarized microglia toward the M2 phenotype, which effectively inhibited the expression of inflammatory factors in SI mice. Finally, the protective effect of betaine treatment in SI mice might not be due to altered activity of the hypothalamic-pituitary-adrenal axis. Collectively, our findings reveal that betaine can improve SI-induced cognitive impairment, thus providing an alternative natural source for the prevention of memory loss caused by SI or loneliness.

Prognostic value of pretreatment systemic immune-inflammation index in Chinese esophageal squamous cell carcinoma patients receiving radical radiotherapy: A meta-analysis.

BACKGROUND: The association between pretreatment systemic immune-inflammation index (SII) and long-term survival among Chinese esophageal squamous cell carcinoma (ESCC) patients who received radical radiotherapy remains unclear. The aim of this study was to identify the prognostic role of pretreatment SII in Chinese ESCC patients receiving radical radiotherapy based on current evidence. METHODS: The PubMed, EMBASE, Web of Science and CNKI databases were searched up to March 18, 2023. Primary and secondary outcomes were overall survival (OS) and progression-free survival (PFS), respectively. The hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) were combined to assess the predictive role of pretreatment SII for long-term survival of Chinese ESCC patients receiving radiotherapy. All statistical analyses were conducted by STATA 15.0 software. RESULTS: A total of 8 eligibility studies involving 2101 cases were included in this meta-analysis. The pooled results demonstrated that elevated pretreatment SII was significantly related to worse OS (HR = 1.59, 95% CI: 1.24-2.02, P < .001) and PFS (HR = 1.33, 95% CI: 1.13-1.57, P < .001). Besides, subgroup based on TNM stage showed similar results. CONCLUSION: Pretreatment SII could serve as a novel prognostic factor in Chinese ESCC patients receiving definitive radiotherapy and patients with an elevated SII may experience poorer survival.

Inflammatory Cytokines Changed in Patients With Depression Before and After Repetitive Transcranial Magnetic Stimulation Treatment.

Studies have found that repetitive transcranial magnetic stimulation rTMS can produce antidepressant effects by affecting inflammatory cytokines in patients with depression, which plays a key role in the therapeutic mechanism of antidepressants. This study aimed to explore the changes in inflammatory cytokine levels in patients with depression after 4 weeks of rTMS treatment to determine the possible antidepressant mechanism of rTMS. This prospective, double-blind, pseudo-stimulus-controlled study was conducted, and a total of 57 patients with depression and 30 healthy controls were recruited. Patients were randomly divided into the active rTMS (n = 29) and sham rTMS groups (n = 28). The Hamilton Depression Scale was used to evaluate depressive symptoms and their severity. The serum levels of seven inflammatory cytokines were measured using enzyme-linked immunosorbent assay. Inflammatory cytokines include high-sensitivity C-reactive protein (CRP-hc); tumor necrosis factor (TNF-α); interferon (IFN-γ); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-6 (IL-6); and interleukin-8 (IL-8). At baseline, TNF-α (F = 36.699, p < 0.001), IFN-γ (F = 8.907, p < 0.001), IL-4 (F = 66.256, p < 0.001), and IL-2 (F = 9.162, p < 0.001) levels in the depression group were significantly different from those of healthy controls. In the self-control analysis of the active rTMS group, the levels of IL-2 and CRP-hc increased significantly after 2 and 12 weeks of treatment. In the sham-rTMS group, IFN-γ increased after 2 and 12 weeks of treatment. Our results revealed that the changes in inflammatory cytokines after rTMS treatment showed different patterns compared to the sham group, suggesting that the antidepressant effect of rTMS may be related to changes in inflammatory cytokines.

A new peptide originated from amphibian skin alleviates the ultraviolet B-induced skin photodamage.

Although amphibian-derived bioactive peptides have attracted increasing attention for their potential use in the treatment of photodamage, research is still in its infancy. In this study, we obtained a new antioxidant peptide, named OA-GI13 (GIWAPWPPRAGLC), from the skin of the odorous frog Odorrana andersonii and determined its effects on ultraviolet B (UVB)-induced skin photodamage as well as its possible molecular mechanisms. Results showed that OA-GI13 directly scavenged free radicals, maintained the viability of hydrogen peroxide-challenged keratinocytes, promoted the release of superoxide dismutase, catalase, and glutathione, and reduced the level of lactate dehydrogenase. Furthermore, topical application of OA-GI13 in mice alleviated dorsal skin erythema and edema and protected the skin against UVB irradiation by increasing antioxidant levels and decreasing peroxide, malondialdehyde, and 8-hydroxydeoxyguanosine levels. OA-GI13 also alleviated oxidative stress injury in vivo and in vitro, possibly by inhibiting p38 protein phosphorylation. Our study confirmed the anti-photodamage effects of this novel amphibian-derived peptide, thus providing a new molecule for the development of drugs and topical agents for the treatment of skin photodamage.

TRPV4 inhibitor HC067047 produces antidepressant-like effect in LPS-induced depression mouse model.

Inflammation is a crucial component that contributes to the pathogenesis of major depressive disorder. It has been revealed that the nonselective cation channel transient receptor potential vanilloid 4 (TRPV4) profoundly affects a variety of physiological processes, including inflammation. However, its roles and mechanisms in LPS-induced depression are still unclear. Here, for the first time, we found that there was a significant increase in TRPV4 in the hippocampus in a depression mouse model induced by LPS. TRPV4 inhibitor HC067047 or knockdown the hippocampal TRPV4 with TRPV4 shRNA could effectively rescue the aberrant behaviors. Furthermore, TRPV4 inhibitor HC067047 reduced the activation of astrocyte and microglia, decreased expression of CaMKII-NLRP3 inflammasome and increased the expression of neurogenesis marker DCX in the hippocampus. In addition, enhanced neuroinflammation in the serum was also reversed by TRPV4 inhibitor HC067047. Thus, we consider that TRPV4 has an important role in contributing to the depression-like behavior following LPS-induced systemic inflammation.

Roles of physical exercise in neurodegeneration: reversal of epigenetic clock.

The epigenetic clock is defined by the DNA methylation (DNAm) level and has been extensively applied to distinguish biological age from chronological age. Aging-related neurodegeneration is associated with epigenetic alteration, which determines the status of diseases. In recent years, extensive research has shown that physical exercise (PE) can affect the DNAm level, implying a reversal of the epigenetic clock in neurodegeneration. PE also regulates brain plasticity, neuroinflammation, and molecular signaling cascades associated with epigenetics. This review summarizes the effects of PE on neurodegenerative diseases via both general and disease-specific DNAm mechanisms, and discusses epigenetic modifications that alleviate the pathological symptoms of these diseases. This may lead to probing of the underpinnings of neurodegenerative disorders and provide valuable therapeutic references for cognitive and motor dysfunction.

A novel amphibian-derived peptide alleviated ultraviolet B-induced photodamage in mice.

Although the application potential of amphibian skin-derived active peptides in alleviating ultraviolet B (UVB)-induced damage has attracted increasing attention, research remains in its infancy. In this study, a new peptide (OM-GL15, GLLSGHYGRASPVAC) was identified from the skin of the green odorous frog (Odorrana margaretae). Results showed that OM-GL15 scavenged free radicals (2,2'-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine) and reduced Fe3+ to Fe2+. Moreover, topical administration of OM-GL15 significantly alleviated UVB-induced skin photodamage in mice. Exploration of the underlying mechanisms further showed that OM-GL15 exerted antioxidant potency. Specifically, the peptide reduced the levels of lipid peroxidation and malondialdehyde and protected epidermal cells from UVB-induced apoptosis by inhibiting DNA damage via down-regulation of p53, caspase-3, caspase-9, and Bax and up-regulation of Bcl-2. Our results highlight the potential application of amphibian skin-derived peptides in protection against UVB-induced photodamage and provide a novel peptide candidate for the development of anti-photodamage agents.

Endocrine Dysfunction Following Stroke.

Endocrine dysfunction is known to occur after traumatic brain injury. The purpose of this study was to examine the incidence of various endocrine dysfunctions after a stroke. The Taiwan National Health Insurance Research Database (NHIRD) was searched from 2001 to 2011 for patients with a diagnosis of stroke. Stroke patients were matched by diagnosis date, age, and sex to patients without a stroke. Cox proportional hazards regression analyses were performed to compare the incidence of goiter, acquired hypothyroidism, thyroiditis, pituitary dysfunction, and disorders of the adrenal glands between stroke and non-stroke patients. There were 131,951 patients in the stroke group, and 131,951 in the matched non- stroke group (mean age 66.1 ± 14.9 years). Stroke patients had significantly higher risk of acquired hypothyroidism (crude hazard ratio [cHR] = 1.65, 95% confidence interval [CI]: 1.44, 1.90; adjusted hazard ratio [aHR] = 1.65, 95% CI: 1.42, 1.91), pituitary dysfunction (cHR = 2.32, 95% CI: 1.79, 2.99; aHR = 1.92, 95% CI: 1.46, 2.52), and disorders of the adrenal glands (cHR = 1.79, 95% CI: 1.52, 2.12; aHR =1.62, 95% CI: 1.36, 1.92) than non-stroke patients. Pituitary dysfunction and disorders of the adrenal glands were found in both hemorrhagic stroke and ischemic stroke patients, while hypothyroidism was seen in ischemic stroke patients only. No significant association was found for goiter and thyroiditis. In conclusions, stroke survivors have an approximately 2-fold increased risk of developing acquired hypothyroidism, pituitary dysfunction, or disorders of the adrenal glands. These risks should be taken into account in the management of patients who have ischemic or hemorrhagic strokes. Graphical Abstract.

Effect and Molecular Mechanisms of Collateral Vessel Growth Mediated by Activation of Transient Receptor Potential Vanilloid Type 1.

Information on the function of transient receptor potential vanilloid 1 (TRPV1) in arteriogenesis is limited. We aimed to verify whether TRPV1 is involved in collateral vessel growth in rat hind limbs and elucidate the possible subcellular action mechanisms. Adult Sprague Dawley rats were chosen to establish the hind limb ischemic model and treatment with capsaicin. Angiographies were performed, and tissue was isolated for immunohistochemistry. In vitro, rat aortic endothelial cells (RAECs) were treated with capsaicin and antagonist capsazepine. The RAEC proliferation was determined, and the protein and mRNA levels of Ca2+-dependent transcription factors were assessed. In vivo, the collateral vessels exhibited positive outward remodeling characterized by enhanced inflammatory cell/macrophage accumulation in the adventitia and activated cell proliferation in all layers of the vascular wall and elevated endothelial NO synthetase expression in the rats with hind limb ligation. In RAECs, TRPV1 activation-induced Ca2+-dependent transcriptional factors, nuclear factor of activated T cells 1, calsenilin and myocyte enhancer factor 2C increase, and augmented RAEC proliferation could be a subcellular mechanism for TRPV1 in endothelial cells and ultimately contribute to collateral vessel growth. TRPV1, a novel candidate, positively regulates arteriogenesis, meriting further studies to unravel the potential therapeutic target leading to improved collateral vessel growth for treating ischemic diseases.

Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii.

With increasing demand, the development of new natural antioxidants has become a primary direction of scientific research. We previously identified a short gene-encoded peptide (OA-VI12) from Odorrana andersonii frog skin secretions that exerted direct scavenging capacity against free radicals, suggesting a possible function in protecting skin against photodamage caused by their high-altitude habitat. In the current research, we examined the effects of OA-VI12 on both UVB-irradiation and hydrogen peroxide-induced oxidative stress models established with human immortalized keratinocytes. In addition, we identified the differentially expressed genes (DEGs) in the oxidative stress and OA-VI12 groups and further performed transcriptome as well as functional and pathway enrichment analyses. Results showed that OA-VI12 protected cell viability, promoted the release of catalase, and decreased the levels of lactate dehydrogenase and reactive oxygen species. Moreover, the peptide promoted the production of superoxide dismutase and glutathione, alleviated epidermis and dermis thickness, and decreased the production of light spots and collagen fibers in skin from the photo-injured mouse model. Kyoto Encyclopedia of Genes and Genomes analysis showed mitogen-activated protein kinase (MAPK) to be the most abundant signaling pathway. Gene Ontology (GO) analysis indicated that the top 55 significantly enriched GO terms mainly involved cellular processes, parts, and binding. These results revealed the beneficial role of the small molecule gene-encoding antioxidant peptide (OA-VI12) and its potential application as a protective agent against photodamage.

A short peptide potentially promotes the healing of skin wound.

Skin wound, a common form of skin damage in daily life, remains a serious challenge in clinical treatment. Bioactive peptides with high efficiency have been considered as potential therapeutic candidates for wound healing. In this report, a novel short linear peptide, with mature peptide sequence of 'GLLSGINAEWPC' and no obvious similarity with other known bioactive peptides, was identified by genomic method from the skin of odorous frog, Odorrana andersonii Our results suggested that OA-GL12 (OA: abbreviation of species (O. andersonii), GL: two initial amino acids, 12: peptide length) obviously accelerated the scratch-healing of keratinocytes and human fibroblasts in a time- and concentration-dependent manner. Meanwhile, OA-GL12 showed significant effect in promoting the wound healing on the full-thickness skin wound model. Inflammatory assay results demonstrated that OA-GL12 induced the secretion of tumor necrosis factor (TNF) and transforming growth factor ß1 (TGF-ß1) on murine macrophage cell line (RAW264.7), which might explain the powerful accelerating capacity of wound healing. Moreover, results also indicated that epidermal growth factor receptor (EGFR) was involved in the mechanisms underlying the scratch-healing promoting activity of OA-GL12. In addition, OA-GL12 showed obvious free radical scavenging activity. Results supported that OA-GL12 did not exert risk in acute toxicity, hemolytic activity, and direct antibacterial activity. The remarkable effect of OA-GL12 on promoting wound healing verified in this research made it potential to be a novel template for the development of wound healing-promoting agents.

The Neuropeptide Secretoneurin Exerts a Direct Effect on Arteriogenesis In Vivo and In Vitro.

It is well known that nerves modulate the development and remodeling of blood vessels by releasing different neuropeptides and neurotransmitters. Secretoneurin (SN), a neuropeptide located in nerve fibers along blood vessels, acts as a pro-angiogenic agent and induces postnatal vasculogenesis. However, little is known about its involvement in arteriogenesis. In the present study, we tested the hypothesis that SN promotes arteriogenesis in a rat model of hind limb ischemia, as such, we evaluated the effect of this neuropeptide on proliferation and the production of adhesion and chemotaxis molecules in vascular smooth muscle cells (VSMCs), the main component that carries the burden of the transformation of a small arteriole into a large collateral vessel. In vivo, SN-immunoreactive nerve fibers were abundantly distributed in the adventitia of the collateral vessel. Moreover, administration of SN induced cell proliferation in the vascular wall and the infiltration of inflammatory cells/macrophages to promote collateral vessel growth. This was shown by an increased density of arterioles/arteries, together with a well-developed network of collateral vessels, and well-preserved skeletal muscles. In vitro, SN exerted proliferative effects on VSMCs and stimulated these cells to express adhesion molecules. In conclusion, our data demonstrate for the first time that SN acts as a mediator of inflammation, contributing to collateral vessel growth, in addition to directly stimulating cell proliferation in the vascular wall to promote collateral vessel growth in a rat model of hind limb ischemia. Anat Rec, 301:1917-1927, 2018. © 2018 Wiley Periodicals, Inc.

Expression of vimentin and glial fibrillary acidic protein in central nervous system development of rats.

OBJECTIVE: To investigate the distribution and contents of vimentin (Vim) and glial fibrillary acidic protein (GFAP) immunoreactivities in the central nervous system (CNS) of normal newborn, adult and aged rats. METHODS: In this study, thirty healthy and normal Sprague-Dawley rats were simply classified into three groups: Newborn (7 days aged), adult (5 months aged) and aged (24 months aged) rats. Brains and spinal cord were dissected and cut into frozen sections. The expression of Vim and GFAP in CNS were detected by confocal immunofluorescence. RESULTS: In each group, Vim was expressed in all the regions of CNS including the hippocampal, cerebral cortex, the third ventricle and spinal cord, and the expression was highest in neuron-like cell of newborn rats, while Vim was mainly expressed in cell bodies in adult and aged rats. GFAP was expressed in all the regions of CNS including the hippocampal, cerebral cortex, the third ventricle and spinal cord, and the expression was in astrocytes of aged rats. In the third ventricle, Vim was detected in all groups, and only observed in neuron-like cells of newborn. Meanwhile, the GFAP expression showed no significant differences between adult and aged rats in this region. The co-localization of Vim and GFAP were mainly observed in hippocampus and cerebral cortex of newborn, but this co-localization was found in the third ventricle of the rats in all groups. CONCLUSION: Our data demonstrate for the first time that the expression of Vim and GFAP in the rat's CNS during development. This data may provide a foundation for the further mechanistic studies of these two main intermediate filaments during development of CNS.

Effect of shunting of collateral flow into the venous system on arteriogenesis and angiogenesis in rabbit hind limb.

The aim of this study was to characterize the vascular remodeling in the external iliac artery (EIA) and the lower leg muscles in a rabbit shunt model created between the distal stump of the occluded femoral artery and the accompanying vein. Histology and immunoconfocal microscopy were used in this study. We found that: 1) both endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (P-eNOS) proteins were significantly increased in the shunt-side EIA; 2) matrix metalloproteinase-2 (MMP-2) expression was 5.5 times in shunt side EIA over that in normal EIA; 3) intercellular adhension molecule-1 (ICAM-1) expression was strongly induced in endothelial cells (EC) and vascular adhension molecule-1 (VCAM-1) expression was significantly increased in both EC and the adventitia of the shunt-side EIA; 4) augmentation of cell proliferation and extracellular proteolysis by macrophage infiltration was observed in shunt-side EIA; 5) cell proliferation was active in shunt side EIA, but quiet in shunt side lower leg's arterial vessels; 6) capillary density in shunt side lower leg muscles was 2 times over that in normal side. In conclusion, our data demonstrate the paradigm that the power of shear stress takes the reins in arteriogenesis, whereas ischemia in angiogenesis, but not in arteriogenesis.

Actin-binding Rho activating protein is expressed in the central nervous system of normal adult rats.

Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.

Comparative proteome analysis of 3T3-L1 adipocyte differentiation using iTRAQ-coupled 2D LC-MS/MS.

Adipose tissue is critical in obesity and type II diabetes. Blocking of adipocyte differentiation is one of the anti-obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Therefore, we exposed 3T3-L1 cell line to appropriate hormonal inducers as adipocyte differentiation model. Using iTRAQ-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, we nearly quantitated 1,000 protein species and found 106 significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules, more than 20 altered proteins previously unknown to be involved with adipogenic process were firstly revealed (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, TPP1, etc.). The partially differential proteins were verified by Western blot and/or real-time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method, and the results showed that they could contribute considerably to adipogenesis. In conclusion, our data provide valuable information for further understanding of adipogenesis.

Collateral vessel growth induced by femoral artery ligature is impaired by denervation.

Innervation plays an important role in development and remodeling of blood vessels. However, very little is known whether innervation is involved in arteriogenesis. In the present study, we tested the hypothesis that innervation may contribute to the process of arteriogenesis induced by ligature of femoral artery in rat/rabbit hind limb with or without denervation. We found that: (1) angiography showed more collateral vessels in the ligature side than that in ligature plus denervation side; (2) collateral vessels in denervation side was characterized by an inward remodeling; (3) in both collateral vessels (CVs) from only femoral ligature side as well as the ligature plus denervation side, ICAM-1 and VCAM-1 expression was up-regulated but increased VCAM-1 was more evident in the adventitia of collateral vessels of only femoral ligature side; (4) 7 days after surgery, in CVs from the femoral ligature side only, numerous macrophages (RAM11 positive cells) and high cell proliferation ratio (ki67 positive cells) were detected, but they were less in the denervation side. In conclusion, our data demonstrate for the first time that neural regulation is one of the factors that contributes to collateral vessel growth in rat/rabbit hind limb ischemic model by showing collateral vessel growth induced by femoral artery ligature is impaired by denervation.

Activation of the integrins alpha 5beta 1 and alpha v beta 3 and focal adhesion kinase (FAK) during arteriogenesis.

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.