Targeted disruption of the Fgf2 gene does not affect vascular growth in the mouse ischemic hindlimb.

Ischemic revascularization involves extensive structural adaptation of the vasculature, including both angiogenesis and arteriogenesis. Previous studies suggest that fibroblast growth factor (FGF)-2 participates in both angiogenesis and arteriogenesis. Despite this, the specific role of endogenous FGF-2 in vascular adaptation during ischemic revascularization is unknown. Therefore, we used femoral artery ligation in Fgf2(+/+) and Fgf2(-/-) mice to test the hypothesis that endogenous FGF-2 is an important regulator of angiogenesis and arteriogenesis in the setting of hindlimb ischemia. Femoral ligation increased capillary and arteriole density in the ischemic calf in both Fgf2(+/+) and Fgf2(-/-) mice. The level of angiographically visible arteries in the thigh was increased in the ischemic hindlimb in all mice, and no significant differences were observed between Fgf2(+/+) and Fgf2(-/-) mice. Additionally, limb perfusion progressively improved to peak values at day 35 postsurgery in both genotypes. Given the equivalent responses observed in Fgf2(+/+) and Fgf2(-/-) mice, we demonstrate that endogenous FGF-2 is not required for revascularization in the setting of peripheral ischemia. Vascular adaptation, including both angiogenesis and arteriogenesis, was not affected by the absence of FGF-2 in this model.     

Protease-Activated Receptor (PAR)2, but Not PAR1, Is Involved in Collateral Formation and Anti-Inflammatory Monocyte Polarization in a Mouse Hind Limb Ischemia Model

Aims

In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model.

Methods and Results

PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered.

Conclusion

PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.     

Nitrite anion stimulates ischemic arteriogenesis involving NO metabolism

Nitric oxide (NO) is a potential regulator of ischemic vascular remodeling, and as such therapies augmenting its bioavailability may be useful for the treatment of ischemic tissue diseases. Here we examine the effect of administering the NO prodrug sodium nitrite on arteriogenesis activity during established tissue ischemia. Chronic hindlimb ischemia was induced by permanent unilateral femoral artery and vein ligation. Five days postligation; animals were randomized to control PBS or sodium nitrite (165 μg/kg) therapy twice daily. In situ vascular remodeling was measured longitudinally using SPY angiography and Microfil vascular casting. Delayed sodium nitrite therapy rapidly increased ischemic limb arterial vessel diameter and branching in a NO-dependent manner. SPY imaging angiography over time showed that nitrite therapy enhanced ischemic gracillis collateral vessel formation from the profunda femoris to the saphenous artery. Immunofluorescent staining of smooth muscle cell actin also confirmed that sodium nitrite therapy increased arteriogenesis in a NO-dependent manner. The NO prodrug sodium nitrite significantly increases arteriogenesis and reperfusion of established severe chronic tissue ischemia.     

Enhanced arteriogenesis in mice overexpressing erythropoietin

After permanent occlusion of the femoral artery, the survival of ischemic limb tissue depends on collateral artery growth (arteriogenesis). In previous work, we have shown that shear stress triggers arteriogenesis. To test whether increased shear stress results in enhanced arteriogenesis, we compared arteriogenesis in transgenic mice overexpressing erythropoietin (EPO), which possessed increased blood viscosity through the higher hematocrit (thereby providing increased shear stress), with wild-type mice. The right femoral artery was occluded proximal to the origin of the arteria poplitea. Distal blood flow was assessed by laser Doppler imaging, and the growth and remodeling of collateral arteries was examined by light and electron microscopy and morphometry. After occlusion of the femoral artery, EPO mice demonstrated enhanced arteriogenesis: their collateral arteries developed a 1.7-fold diameter and a 2-fold wall thickness compared with wild-type. However, the blood flow recovery in EPO mice was markedly retarded. Structural remodeling and growth of collateral arteries was markedly enhanced in EPO mice, presumably as a result of increased blood viscosity and shear stress.     

MiR-335 Regulates Hif-1α to Reduce Cell Death in Both Mouse Cell Line and Rat Ischemic Models

Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.     

Enhanced expression of the Flt‐1 and Flk‐1 receptor tyrosine kinases in a newborn piglet model of ischemic tolerance

Vascular endothelial growth factor (VEGF), an angiogenic factor induced by hypoxia, also exerts direct effects on neural tissues. VEGF up‐regulation after hypoxia coincides with expression of its two tyrosine kinase receptors Flt‐1(VEGFR‐1) and Flk‐1 (KDR/VEGFR‐2), which are the key mediators of physiological angiogenesis. We have recently shown that hypoxic‐preconditioning (PC) leading to tolerance to hypoxia–ischemia in neonatal piglet brain resulted in increased expression of VEGF. In this study, we used a hypoxic‐preconditioning model of ischemic tolerance to analyze the expression and cellular distribution of VEGF receptors and phosphorylation of cAMP‐response element‐binding protein (CREB) in newborn piglet brain. The response of Flt‐1 and Flk‐1 mRNA to PC alone was biphasic with peaks early (6 h) and late (1 week) after PC. The mRNA expression of Flt‐1 and Flk‐1 in piglets preconditioned 24 h prior to hypoxia–ischemia was significantly higher than non‐preconditioned piglets and remained up‐regulated up to 7 days. Furthermore, PC prior to hypoxia–ischemia significantly increased the protein levels of Flt‐1 and Flk‐1 compared with hypoxia–ischemia in a time‐dependent manner. Double‐immunolabeling indicated that both Flt‐1 and Flk‐1 are expressed in neurons and endothelial cells with a similar time course of expression following PC and that PC leads to the growth of new vessels. Finally, our data demonstrate that PC significantly phosphorylated and activated cAMP‐response element‐binding protein in nucleus. These results suggest that mechanism(s) initiated by PC can induce VEGF receptor up‐regulation in newborn brain and that VEGF–VEGF receptor‐coupled signal transduction pathways could contribute to the establishment of tolerance following hypoxia–ischemia.     

Anthrax toxin receptor 2 functions in ECM homeostasis of the murine reproductive tract and promotes MMP activity

Anthrax Toxin Receptor proteins function as receptors for anthrax toxin, however physiological activity remains unclear. To evaluate the biological role of Antxr2, we generated Antxr2-/- mice. Antxr2-/- mice were viable, however Antxr2 is required for parturition in young females and for preserving fertility in older female mice. Histological analysis of the uterus and cervix revealed aberrant deposition of extracellular matrix proteins such as type I collagen, type VI collagen and fibronectin. A marked disruption of both the circular and longitudinal myometrial cell layers was evident in Antxr2-/- mice. These changes progressed as the mice aged, resulting in a thickened, collagen dense, acellular stroma and the disappearance of normal uterine architecture. To investigate the molecular mechanism underlying the uterine fibrosis we performed immunoblotting for MMP2 using uterine lysates and zymography using conditioned medium from Antxr2-/- mouse embryonic fibroblasts and found reduced levels of activated MMP2 in both. This prompted us to investigate MT1-MMP status, as MMP2 processing is regulated by MT1-MMP. We found MT1-MMP activity, as measured by MMP2 processing and activation, was enhanced by expression of either ANTXR1 or ANTXR2. We identified an ANTXR2/MT1-MMP complex and demonstrated that MT1-MMP activity is dependent on ANTXR2 expression levels in cells. Thus, we have discovered that ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity.     

Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.     

缺血缺氧再灌注对胎鼠肾组织环氧化酶表达的影响

目的观察宫内缺血缺氧再灌注后,胎鼠肾组织COX蛋白表达及其AA代谢产物PGI2、PGE2和TXA2含量的变化,探讨COX在宫内窘迫胎鼠肾损伤发病机制中的作用.方法制备胎鼠子宫内缺血缺氧再灌注模型(缺血缺氧组：缺血缺氧30min;再灌注组：缺血缺氧30min后,分别再灌注30min,2h,6h,12h,24h,30h)缺血氧组：取胎鼠18只;再灌注组各时间点分别取胎鼠18只;对照组取胎鼠20只.将肾组织匀浆后采用Western免疫印迹和放免法检测COX蛋白表达及其PGI2、PGE2和TXA2含量的变化.同时HE染色观察肾组织病理学改变.结果子宫内缺血缺氧再灌注后胎肾组织COX-2蛋白表达上调,PGI2的稳定代谢产物6-Keta-PGF1α及PGE2均于再灌注2h开始增高(P<0.05).其中6-keta-PGF1α增加迅速,于再灌注12h达假手术组的6倍(P<0.01),PGE2于再灌注24h达假手术组的2倍(P<0.01),而TXB2增加幅度不大,无统计学意义.结论宫内缺血缺氧再灌注选择性地诱导胎肾COX-2蛋白表达增强,COX-2可能通过PGI2和PGE2对缺血性胎肾损伤具有保护作用,因此,在围产期肾损伤不宜应用COX-2抑制剂.     

A limited role for regulatory T cells in post-ischemic neovascularization

Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4(+) CD25(+) FoxP3(+) T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro-coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)-2 with the IL-2 monoclonal antibody JES6-1, or depleted by anti-CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post-ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non-depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.     

Detrimental Effect of Class-selective Histone Deacetylase Inhibitors during Tissue Regeneration following Hindlimb Ischemia

Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreated mice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formed muscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereas MS275 inhibited it. Capillarogenesis did not change. Chromatin immunoprecipitations revealed that class IIa HDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues.     

A pharmacological approach to thromboxane receptor antagonism

Thromboxane A2 (TxA2) appears to be an important mediator of ischemia and hypoxia. Despite its short half-life and the fact that it may not circulate in the blood until its values become quite high, TxA2 contributes to the pathogenesis of cardiopulmonary diseases (e.g., sudden death, myocardial ischemia, circulatory shock). It does so because it propagates its own formation by activating platelets and constricting blood vessels, thus activating more TxA2 and trapping it locally within an ischemic or hypoxic region. TxA2 concentrations in the extracellular fluid of lymph of ischemic regions may be much higher than that occurring in nonischemic, normally perfused regions. Specific and potent Tx receptor antagonists (TxRA) have recently become available for study. The TxRA are useful tools in the study of the pathophysiology of Tx-dependent disease processes and have been found to be effective in a variety of ischemic disorders including circulatory shock, myocardial ischemia, and sudden cardiopulmonary death. Moreover, inasmuch as early work indicates that these agents are both safe and effective in humans, Tx receptor antagonists may be employed as therapeutic agents in several cardiovascular disease states. Further investigation is necessary to clarify the role of TxRA as therapeutic agents.     

Sirt3 Protects Against Ischemic Stroke Injury by Regulating HIF-1α/VEGF Signaling and Blood–Brain Barrier Integrity

Sirtuin 3 (Sirt3) is a member of the Sirtuin family proteins and known to regulate multiple physiological processes such as metabolism and aging. As stroke is an aging-related disease, in this work, we attempt to examine the role and potential mechanism of Sirt3 in regulating ischemic stroke by using a permanent middle cerebral artery occlusion (pMCAO) model in wild type (WT) and Sirt3 knockout (KO) mice, coupled with oxygen glucose deprivation (OGD) experiments in cultured primary astrocytes. Sirt3 deficiency aggravated neuronal cell apoptosis and neurological deficits after brain ischemia. In addition, Sirt3 KO mice showed more severe blood–brain barrier (BBB) disruption and inflammatory responses compared with WT group in the acute phase. Furthermore, specific overexpression of Sirt3 in astrocytes by injecting glial fibrillary acidic protein (GFAP)::Sirt3 virus in ischemic region showed protective effect against stroke-induced damage. Mechanistically, Sirt3 could regulate vascular endothelial growth factor (VEGF) expression by inhibiting hypoxia inducible factor-1α (HIF-1α) signaling after ischemia (OGD). Our results have shown that Sirt3 plays a protective role in ischemic stroke via regulating HIF-1α/VEGF signaling in astrocytes, and reversal of the Sirt3 expression at the acute phase could be a worthy direction for stroke therapy.

     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Effects of intracerebroventricular injection of delta-opioid receptor agonist TAN-67 or antagonist naltrindole on acute cerebral ischemia in rat

This work was performed to determine the role of delta-opioid receptor (DOR) in protection against acute ischemia/reperfusion injury. Transient (1 h) focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (30 nmol, 60 nmol, 200 nmol), DOR antagonist naltrindole (20 nmol, 50 nmol, 100 nmol) or artificial cerebral spinal fluid (aCSF) was injected respectively into the lateral cerebroventricle of the rat 30 min before the induction of brain ischemia. Neurological deficits were assessed by the five-grade system (Longa's methods). The brain infarct was measured by cresyl violet (CV) staining and infarct volume was analyzed by an image processing and analysis system. The expression of DOR was detected by Western blot. The results showed that 60 nmol TAN-67 significantly reduced the infarct volume (P<0.05), attenuated neurological deficits (P<0.05) and tended to increase the expression of about 60 kDa DOR protein (P>0.05), while 100 nmol naltrindole aggravated ischemic damage and decreased about 60 kDa DOR protein expression (P<0.05). These results suggest that DOR activation protects the brain against acute ischemia/reperfusion injury in rat.     

The art of arteriogenesis

The identification of collateral artery growth (arteriogenesis) as the only mechanism to compensate for the loss of an occluded artery forced us to define the mechanisms responsible for this type of vessel growth. To achieve this, a variety of coronary as well as peripheral models of arteriogenesis have been developed. Based on these studies it is obvious that arteriogenesis obeys different mechanisms than angiogenesis, the sprouting of capillaries. Upon occlusion of an artery, the blood flow is redirected into preexisting arteriolar anastomoses that experience increased mechanical forces such as shear stress and circum ferential wall stress. The endothelium of the arteriolar connections is then activated, resulting in an increased release of monocyte-attracting proteins as well as an upregulation of adhesion molecules. Upon adherence and extravasation, monocytes promote arteriogenesis by supplying growth factors and cytokines that bind to receptors that are expressed on vascular cells within a limited time frame. Animal studies evidenced that factors, such as monocyte chemoattractant protein-1, granulocyte-monocyte colony-stimulating factor, or transforming growth factor-β₁, that either attract or prolong the lifetime of monocytes efficiently enhance collateral artery growth, an effect that was seen only to a minor degree after application of a single growth factor. Bone marrow-derived stems cells and endothelial progenitor cells do not incorporate in growing arteries but, rather, function as supporting cells. Complete elucidation of the mechanisms of arteriogenesis may lead to efficacious therapies counteracting the devastating consequences of vascular occlusive diseases.     

脑缺血／再灌流脑微血管内皮细胞ELAM—1和ICAM—1mRNA表达的研究

目的：探讨ELAM-1和ICAM-1在局部脑缺血/再灌流炎性反应过程中的作用。方法：采用厅局级龙线栓堵大脑中动脉造成局部脑缺血/再灌流模型,用RT-PCR方法检测缺血侧脑组织缺血/再灌流不同时间点ELAM-1和ICAM-1mRNA的表达。结果：假手术组脑组织未见ELAM-1和ICAM-1mRNA的表达,手术组非缺血侧脑组织仅见少量表达。脑缺血/再灌流后1h,缺血侧脑组织ELAM-1和ICAM-1mRNA的表达量已开始升高;再灌流后3h,ICAM-1mRNA的上调达高峰,而ELAM-1mRNA的上调在缺血/再灌流后6h达高峰,且持续至缺血/再灌流后48h。结论：EL-AM-1和ICAM-1参与了局部缺血再灌流脑组织损伤的病理过程。二者在白细胞进入缺血区脑组织的病理过程中发挥着重要作用。     

Cardiomyocytes of chronically ischemic pig hearts express the MDR-1 gene-encoded P-glycoprotein.

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.