Endothelial cell surface vimentin binding peptide induces angiogenesis under hypoxic/ischemic conditions

We have previously identified several angiogenic peptides that bind cell surface proteins by screening a phage display peptide library on human umbilical endothelial cells exposed to hypoxic conditions. In this study we describe one of the selected peptides, SP. We found by protein precipitation of endothelial cell lysates that the 12 amino acid SP peptide binds cell surface vimentin. Surprisingly, vimentin was detected on the cell surface of about 30% of intact endothelial cells under both normoxic and hypoxic conditions, as was demonstrated by fluorocytometric analysis on viable cells. The assessment of SP in the induction of angiogenesis was established by a significant increase in endothelial cell proliferation and tube formation under hypoxic conditions and not under normoxic conditions. Cell proliferation and tube length increased two-fold in endothelial cells in the presence of 10 ng/ml SP peptide when compared to controls. The specificity of SP binding to vimentin was demonstrated by SP inhibition of anti-vimentin binding and by the inhibition of tube formation in cells transfected with siRNA against vimentin. Local intramuscular administrations of the peptide SP to ischemic hind limbs using the mouse hind limb ischemia model, demonstrated that SP inoculated at 1 and 10 μg, improved blood perfusion compared to inoculations with an irrelevant peptide or PBS. The recovery of blood perfusion correlated with the increase in the number of detectable capillaries in the ischemic limb. The development of novel peptides for the induction of pro-angiogenic activity may pave the way for new therapeutic strategies in the treatment of cardiovascular ischemic diseases.     

A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic states.

BACKGROUND: Angiogenesis, or new blood vessel formation, is the physiological adaptation of a tissue to hypoxia or ischemia. However, this compensatory response to hypoxic stress in vivo is often insufficient. In many of the conditions in which the angiogenic response to tissue hypoxia is insufficient, such as chronic critical limb ischemia or myocardial hibernation, the hypoxic stress is chronic and persistent, lasting for days or even months. Vascular endothelial growth factor (VEGF) has been demonstrated in vivo and in vitro to be the principal mediator of hypoxia-induced angiogenesis. We propose that the lack of compensatory angiogenesis in response to tissue hypoxia in many clinical syndromes characterized by chronic hypoxia is due to a failure to induce VEGF appropriately. METHODS AND RESULTS: Heart or liver cells were grown under conditions of chronic hypoxia, returned to a normoxic environment, and then rechallenged with hypoxia. We found that the hypoxic induction of VEGF mRNA was markedly blunted using this algorithm. Furthermore, transient transfection studies using the VEGF promoter containing an oxygen-responsive enhancer element failed to show induction in cells pretreated by subjection to chronic hypoxia. CONCLUSIONS: Hypoxic pretreatment results in a blunting of the ability of a cell to induce VEGF in response to subsequent episodes of hypoxia. This may provide a rationale for the inadequate amount of compensatory angiogenesis seen in many chronic ischemic disorders.     

Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair

There is great demand for the development of novel therapies for ischemic cardiovascular disease, a leading cause of morbidity and mortality worldwide. We report here on the development of a completely synthetic cell-free therapy based on peptide amphiphile nanostructures designed to mimic the activity of VEGF, one of the most potent angiogenic signaling proteins. Following self-assembly of peptide amphiphiles, nanoscale filaments form that display on their surfaces a VEGF-mimetic peptide at high density. The VEGF-mimetic filaments were found to induce phosphorylation of VEGF receptors and promote proangiogenic behavior in endothelial cells, indicated by an enhancement in proliferation, survival, and migration in vitro. In a chicken embryo assay, these nanostructures elicited an angiogenic response in the host vasculature. When evaluated in a mouse hind-limb ischemia model, the nanofibers increased tissue perfusion, functional recovery, limb salvage, and treadmill endurance compared to controls, which included the VEGF-mimetic peptide alone. Immunohistological evidence also demonstrated an increase in the density of microcirculation in the ischemic hind limb, suggesting the mechanism of efficacy of this promising potential therapy is linked to the enhanced microcirculatory angiogenesis that results from treatment with these polyvalent VEGF-mimetic nanofibers.     

Retinal endothelial angiogenic activity: effects of hypoxia and glial (Müller) cells

OBJECTIVE: To explore the impact of retinal glial (Müller) cells on survival and neovascularization-related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions. METHODS: Bovine retinal endothelial cells (BRECs) were cultured under normoxia or hypoxia (0.5% O2) either alone, together with the human Müller cell line MIO-M1, or in normoxia- or hypoxia-conditioned media of MIO-M1 cells. Cell number, proliferation, apoptotic cell death, and migration of BRECs were determined. RESULTS: Exposure of BRECs to hypoxia for 24 h decreased the number of adherent cells and the proliferation rate, but increased apoptosis and cell migration. Increased apoptosis and decreased proliferation of the BRECs occurred also in the presence of conditioned media of MIO-M1 cells. Under normoxic conditions, co-culture with MIO-M1 cells resulted in increased proliferation, but decreased apoptosis and migration rates of BRECs. Under hypoxic conditions, the Müller cells released elevated amounts of VEGF but their presence decreased proliferation, apoptosis and the migration rates of BRECs. CONCLUSIONS: Hypoxia inhibits the proliferation of retinal endothelial cells. Müller cells release soluble mediators that enhance this hypoxia-mediated effect but, under certain conditions (i.e., in co-culture), may protect retinal endothelial cells from apoptosis, thus supporting their survival. Altogether the findings indicate that the key signal necessary to trigger retinal endothelial proliferation under hypoxia remains to be determined.     

Transplantation of Cardiac Mesenchymal Stem Cell-Derived Exosomes for Angiogenesis

We demonstrated the effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. To investigate the effect of exosomes from C-MSC on angiogenesis, we injected C-MSC-Exo or PBS intramuscularly into ischemic hind limb. Blood perfusion of limb was evaluated by laser Doppler Imaging. We observed that ischemic limb treated with C-MSC-Exo exhibits improved blood perfusion compared to ischemic limb treated with PBS at 2 weeks and 1 month after induction of limb ischemia. To explore the potential mechanisms underlying C-MSC-Exo’s angiogenetic effect, we performed microRNA array analysis and identify mmu-miR-7116-5p as the most abundant enriched miRNA detected in C-MSC-Exo. Bioinformatics’ analysis shows that miR-7116-5p negatively regulates protein polyubiquitination. In conclusion, our study demonstrated that intramuscular delivery of C-MSC-Exo after limb ischemia improves blood perfusion, and we identified the most abundant miRNAs that are preferentially enriched in C-MSC-Exo.     

Contribution of Bcl-2, but not Bcl-xL and Bax, to antiapoptotic actions of hepatocyte growth factor in hypoxia-conditioned human endothelial cells

Angiogenic growth factors play important roles in angiogenic responses, such as vasculogenesis and angiogenesis in response to hypoxia. A novel angiogenic growth factor, hepatocyte growth factor (HGF), has been reported to inhibit endothelial cell death. However, its molecular mechanisms are largely unknown. Thus, we studied (1) the effects of HGF on hypoxia-induced endothelial apoptosis and (2) the molecular mechanisms of the antiapoptotic actions of HGF in endothelial cells. Severe hypoxia increased the cell death rate in human aortic endothelial cells, whereas HGF significantly attenuated cell death. In addition, hypoxic treatment resulted in a significant increase in apoptotic cells, whereas HGF could attenuate apoptosis, accompanied by attenuation of the increase in caspase-3-like activity (P<0.01). Of importance, HGF significantly increased Bcl-2, an inhibitor of apoptosis, in a dose-dependent manner under normoxic and hypoxic conditions (P<0.01), whereas hypoxic conditions resulted in a significant decrease in Bcl-2. In contrast, HGF failed to affect Bcl-xL, which is also well known as an inhibitor of apoptosis under both normoxic and hypoxic conditions, whereas Bcl-xL was significantly decreased in endothelial cells exposed to hypoxia (P<0.01). No significant change in Bax, a promoter of apoptosis, was also observed in endothelial cells under hypoxia, whereas HGF did not affect BAX: Overall, this study demonstrated that HGF prevented endothelial cell death induced by hypoxia through its antiapoptotic action. The antiapoptotic mechanisms of HGF in hypoxia-induced endothelial cell death largely depend on Bcl-2, but not Bcl-xL and BAX:     

Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle cell mitogen(s)

Bovine pulmonary endothelial cells grown in vitro were shown to release a factor that was mitogenic for pulmonary smooth muscle cells during exposure to a reduced oxygen tension atmosphere. The addition of hypoxic endothelium-derived medium resulted in a 60% increase in smooth muscle cell number after 24 h of exposure. Addition of medium from pulmonary endothelium exposed to normoxic conditions or medium derived from either hypoxic or normoxic aortic endothelium did not result in significant increases in smooth muscle cell number. Physicochemical characterization of the hypoxic pulmonary endothelial cell-derived factor(s) showed that it was resistant to heat and reducing agent treatment and stable between pH 3 and 10. The mitogenic activity decreased by 71% at pH 2 and by 68% after treatment with trypsin. The activity adhered to DEAE Sephadex. Gel filtration chromatography of the hypoxic-conditioned medium demonstrated 2 major peaks of smooth muscle cell growth factor activity corresponding to molecular weights between 6,000 and 14,000 and 20,000 and 65,000 daltons, respectively. These data suggest that this pulmonary endothelial cell-derived smooth muscle cell peptide mitogen(s) may be involved in the smooth muscle cell proliferative response seen with chronic alveolar hypoxia.     

Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms

We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced >1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC(CM)). MSC(CM) promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC(CM) promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC(CM) enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.     

Adenosine exacerbates ischemic myocardial injury during regional coronary hypoxemia in the dog

To investigate the myocardial protective effect of adenosine (ADO), the left anterior descending artery of 10 dogs was cannulated and perfused at a pressure of 100 mmHg. Hypoxic coronary perfusion was conducted for 60 min followed by 60 min of normoxic reperfusion (group 1, control, n = 5). In group 2 (n = 5), ADO was continuously infused at a rate of 40 micrograms/kg/min for 30 min before and during 60 min of hypoxic blood perfusion and for 60 min following normoxic reperfusion. In group 1, R wave amplitude and TQ segment of the epicardial surface electrocardiogram (EPIECG) were reduced, the ST segment was elevated, and a new Q wave developed during hypoxic blood perfusion. However, at 60 min of normoxic reperfusion, the R wave amplitude recovered to approximately 60% of its control value. Also, ST segment elevation was restored to normal and the new Q wave disappeared. The TQ segment returned to normal after approximately 20 min of hypoxic perfusion. Histological analysis showed myocardial infarction in the subendocardium, but not in the subepicardium. In group 2, EPIECG findings during hypoxic blood perfusion were similar to those observed in group 1. However, the recovery of ST segment elevation during normoxic reperfusion was slower than that observed in group 1. R wave restoration did not occur in group 2. Furthermore, the new Q wave remained present throughout the normoxic reperfusion period. Transmural infarction and severe congestion were observed histologically in this group. Obvious contraction bands were not demonstrated histologically in either group. These findings suggest that ADO did not protect myocardial viability during 60 min of hypoxic blood perfusion, but instead, ADO exacerbated ischemic injury during regional hypoxic perfusion.     

匹他伐他汀促进小鼠血管新生作用及其机制的研究

目的探讨匹他伐他汀对小鼠血管新生的促进作用及其可能的作用机制。方法建立野生型C3H／He小鼠下肢缺血模型并分为2组,缺血对照组,匹他伐他汀组。使用激光多普勒血流测定仪测定实验小鼠投药前、下肢缺血手术后双下肢血流。免疫荧光组化sP法计数缺血肢毛细血管数。免疫酶组化直接法计数缺血肢磷酸化蛋白激酶Akt（p-Akt）阳性细胞数。蛋白印迹杂交方法检测缺血肢血管内皮生长因子蛋白表达。镉还原Griess法测定实验结束后血清一氧化氮代谢产物含量。结果匹他伐他汀使实验小鼠术后缺血肢血流恢复明显,缺血肢与非缺血肢血流面积比明显增加;缺血肢毛细血管密度明显增加、p-Akt活性增加（p-Akt阳性细胞数明显增加）;血中一氧化氮代谢产物含量明显增高;缺血肢血管内皮生长因子蛋白表达增强。结论匹他伐他汀有促进小鼠血管新生的作用。     

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. 2000;96:2775-2783)     

Conditioned medium derived from mesenchymal stem cells overexpressing HPV16 E6E7 dramatically improves ischemic limb

Mesenchymal stem cells (MSCs) have been shown to secrete cytokines and growth factors required for angiogenesis. Previously, we demonstrated that MSCs expressing HPV16 E6E7 mRNA (E6E7-MSCs) increase life span and differentiation potential and maintain without neoplastic transformation. Whether E6E7-MSCs are sources of molecules for enhancing angiogenesis is unknown. We demonstrated that E6E7-MSC-derived conditioned medium (E6E7-CM) enhanced endothelial cell migration and tube formation compared to primary MSC-derived conditioned medium (primary-CM). Moreover, E6E7-MSCs increased AKT activation and enhanced the release of Interleukin-1β (IL-1β) and vascular endothelial growth factor A (VEGFA). Neutralization of E6E7-CM with antibodies against IL-1β or VEGFA abrogated its effect in enhancing endothelial migration and tube formation. Primary-CM, added with IL-1β and VEGFA, enhanced its ability to increase endothelial migration and tube formation. E6E7-CM was shown to increase the ability to improve blood perfusion in a mouse limb ischemia model. Histological analysis revealed that E6E7-CM prohibited muscle loss or fibrosis and increased endothelial cell counts compared to primary-CM. Similarly, the effects of E6E7-CM in improving perfusion in ischemic limb were also contributed by the increase of IL-1β or VEGFA levels. These results suggest that E6E7-MSCs increase the ability to secrete angiogenic factors via AKT activation, and E6E7-CM is abundant in IL-1β and VEGFA levels and thereby increases the ability to improve blood perfusion and prohibit muscle loss or fibrosis in a mouse limb ischemia model.     

Matrix metalloprotein-9 activation under cell-to-cell interaction between endothelial cells and monocytes: possible role of hypoxia and tumor necrosis factor-α

Matrix metalloproteinase (MMP)-9 plays an important role in cardiovascular events. However, the mechanisms underlying in vivo activation of MMP-9 are largely unknown. We investigated the secretion and activation of MMP-9 under a cell-to-cell interaction, and the effects of hypoxia and cytokine. Human umbilical vein endothelial cell (HUVEC) and THP-1 (human monocyte cell line) were cultured individually, or cocultured under normoxic and hypoxic conditions. In a coculture of HUVEC and THP-1, proMMP-9 secretion was increased twofold compared with individual culture of HUVEC and THP-1, whereas MMP-2 secretion was unchanged. The increase in proMMP-9 secretion was suppressed by antiadhesion molecule antibodies and mitogen-activated protein kinase inhibitors, PD98059 (MAPK/ERK kinase1 inhibitor) and SP600125 (Jun N-terminal kinase inhibitor). ProMMP-9 secretion was increased by tumor necrosis factor (TNF)-α at 50 ng/ml (P < 0.05) but was not activated under normoxic (20%) conditions. ProMMP-9 in coculture was activated under hypoxic (<1%) conditions, and was potentiated by TNF-α (both P < 0.05). To further investigate the mechanism of hypoxia-induced MMP-9 activation, heat shock protein (Hsp)90, which was suggested to be related to MMP-9 activation, was measured by Western blot analysis. The ratio of Hsp90 to glyceraldehyde-3-phosphate dehydrogenase was increased in hypoxic (<1%) coculture conditions with TNF-α (P < 0.05). Treatment with geldanamycin and 17-DMAG (Hsp90 inhibitor) suppressed the active form of MMP-9. Cell-to-cell interaction between endothelial cells and monocytes promotes proMMP-9 synthesis and secretion. Hypoxia and inflammation are suggested to play an important role in activating proMMP-9, presumably via Hsp90.     

Hyperbaric oxygen induces basic fibroblast growth factor and hepatocyte growth factor expression, and enhances blood perfusion and muscle regeneration in mouse ischemic hind limbs.

BACKGROUND: It is not clear how hyperbaric oxygen therapy (HBO) affects ischemia-induced pathophysiological responses such as angiogenesis and skeletal muscle regeneration. In the present study the effects of HBO on the functional and morphological recovery of ischemic hind limbs, blood perfusion and the local production of angiogenic growth factors were studied in a mouse model. METHODS AND RESULTS: Mice were placed in pure oxygen under 3 atm for 1 h/day for 14 days after the removal of a segment of the left femoral artery. HBO-treated mice showed better functional recovery and greater blood flow in the ischemic hind limb than untreated mice. Histological examination revealed unatrophied muscle fibers with islands of small regenerating muscle cells only in HBO-treated mice. Regeneration of muscle was confirmed by the increase in myf5 mRNA. The amount of mRNA for vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was slightly increased in the ischemic hind limbs. HBO eliminated the increase in VEGF mRNA. In contrast, the amount of mRNA for bFGF and HGF was further increased by HBO treatment. HBO transiently increased early growth response protein 1 (Egr-1) in the ischemic hind limbs. CONCLUSIONS: HBO accelerates the recovery of ischemic hind limbs by increasing the production of bFGF and HGF and by promoting muscle regeneration in mice.     

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.