Chronic Tissue Ischemia Research Articles

Long noncoding RNA LEENE promotes angiogenesis and ischemic recovery in diabetes models.

Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long noncoding RNAs (lncRNAs), are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression was decreased in diabetic conditions in cultured endothelial cells (ECs), mouse hind limb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduced their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in Leene demonstrated impaired angiogenesis and perfusion following hind limb ischemia. Importantly, overexpression of human LEENE rescued the impaired ischemic response in Leene-knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promoted transcription of proangiogenic genes in ECs, such as KDR (encoding VEGFR2) and NOS3 (encoding eNOS), potentially by interacting with LEO1, a key component of the RNA polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.

Skin microhemodynamics in patients with obliterating atherosclerosis of lower extremities arteries and diabetes mellitus type 2

Introduction. Chronic tissue ischemia appeared due to obliterating atherosclerosis of lower extremities arteries (OALEA) enhances diabetic microangiopathy making prognosis worse. Due to fundamental biological importance of microcirculation (MC), the study of tissue blood flow in case of this pathology receives huge theoretical value.Purpose of the study was to reveal features of skin MC in patients with diabetes mellitus type II (DM) with OALEA in comparison with the same group of patients without DM.Material and methods. The study involved 111 males with claudication IIB stage and ancle-brachial index (ABI) ≤0.85, which were randomized into two groups equal in age and main clinical and anamnesis data (Gr.1 –without DM (n=77) and Gr.2 – with DM (n=34)). Skin MC was examined using Laser Doppler Flowmetry.Results. Patients with DM had statistically important lowering of blood flow oscillatory amplitude in myogenic (34,5 %) and endothelial (27,8 %) frequency ranges in comparison with the alternative group. Together with the growth of myogenic tone (63,5 [38,6; 123,4] vs 43,6 [28,9; 75,0] u), it reflects depression of vasolytic features and constriction of precapillary segment of the bloodstream. The study revealed significant limitation (18,9 %) of nutritional blood flow and increase of the intensity of arterial and venous bypass grafting in patients of Gr.2 (2,6 [1,8; 4,0 vs 2,0 [1,2; 3,0] u) in Gr. 1.Conclusion. The studied patients had monotype MC alterations. However, they are more pronounced in patients with DM; it worsens the disease prognosis.

Topic: Echocardiographic Evaluation of Left Ventricular Systolic and Diastolic Function in Nigerians with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is chronic, inherited haemoglobin disorder, associated with chronic tissue ischemia which may adversely affect any organ system. Chronic anaemia in SCD results in cardiac chamber dilatation with compensatory increase in left ventricular mass and varying degree of diastolic dysfunction that has been a strong independent predictor of mortality in patients with SCD. There is paucity of echocardiographic studies on adults Nigerians with SCD. This study therefore, aimed to assess left ventricular systolic and diastolic function among sickle cell disease patients in Kano State, North-Western Nigeria. Methods: The study was cross-sectional and comparative conducted at the SCD clinic and Medical/outpatient (MOP) clinic of Murtala Muhammad Specialist Hospital (MMSH), on eligible patients aged 13 years and above. One hundred patients with SCD (HbSS) were recruited as the study group while 100 non SCD (HbAA) patients, matched for age and sex served as controls. Left and right atrial and ventricular dimensions, left ventricular (LV) wall thickness, LV mass index and LV contractility variables were obtained. Parameters of LV diastolic function were also evaluated. Results: There were increases in the left atrial and left ventricular dimensions, left ventricular volumes and left ventricular mass (LVM) of the SCD patients. LV ejection fraction was equivalent, though there was evidence of left ventricular diastolic dysfunction in up to 36%. Conclusion: Left ventricular diastolic dysfunction may complicate cases adults with SCD.

Vascular Remodelling and Mesenchymal Transition in Systemic Sclerosis

Fibrosis of the skin and of internal organs, autoimmunity, and vascular inflammation are hallmarks of Systemic Sclerosis (SSc). The injury and activation of endothelial cells, with hyperplasia of the intima and eventual obliteration of the vascular lumen, are early features of SSc. Reduced capillary blood flow coupled with deficient angiogenesis leads to chronic hypoxia and tissue ischemia, enforcing a positive feed-forward loop sustaining vascular remodelling, further exacerbated by extracellular matrix accumulation due to fibrosis. Despite numerous developments and a growing number of controlled clinical trials no treatment has been shown so far to alter SSc natural history, outlining the need of further investigation in the molecular pathways involved in the pathogenesis of the disease. We review some processes potentially involved in SSc vasculopathy, with attention to the possible effect of sustained vascular inflammation on the plasticity of vascular cells. Specifically we focus on mesenchymal transition, a key phenomenon in the cardiac and vascular development as well as in the remodelling of injured vessels. Recent work supports the role of transforming growth factor-beta, Wnt, and Notch signaling in these processes. Importantly, endothelial-mesenchymal transition may be reversible, possibly offering novel cues for treatment.

The role of molecular imaging in the evaluation of myocardial and peripheral angiogenesis.

Angiogenesis, or the formation of new microvasculature, is a physiological process that may occur in the setting of chronic tissue ischemia and can play an important role in improving tissue perfusion and blood flow following myocardial infarction or in the presence of peripheral vascular disease (PVD). Molecular imaging of angiogenesis within the cardiovascular system is a developing field of study. Targeted imaging of angiogenesis has the potential for non-invasive assessment of the underlying molecular signaling events associated with the angiogenic process and, when applied in conjunction with physiological perfusion imaging, may be utilized to predict and evaluate clinical outcomes in the setting of ischemic heart disease or PVD. This review discusses the developing radiotracer-based imaging techniques and technology currently in use that possess potential for clinical translation, with specific focus on PET and SPECT imaging of myocardial and peripheral angiogenesis.

Hypoxia-inducible factor 1-α in chronic gastrointestinal ischemia

Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic ischemic and inflammatory conditions of the human gastrointestinal (GI) tract. Immunohistochemical expression of HIF-1α was analyzed in 61 patients, including patients with CGI, Helicobacter pylori gastritis, ischemic colitis (IC), infectious colitis, and inflammatory bowel disease (IBD), and 22 controls. HIF-1α expression in >10 % of the cells was regarded as positive staining, and expression <10 % of the cells was considered as negative staining. In the upper GI tract, HIF-1α expression was found in 5/20 CGI patients, but not in controls (p = 0.08). The sensitivity and specificity of HIF-1α expression for diagnosing CGI were 25 and 84%, respectively. In the lower GI tract, HIF-1α was expressed in all patients with IC and infectious colitis and in a majority of IBD patients as well as in 7/12 controls. The sensitivity and specificity of HIF-1α for diagnosing IC were 100 and 51%, respectively. HIF-1α expression was more often (p = 0.02) observed in patients with histological signs of inflammation in the lower GI tract. HIF-1α is expressed in acute and chronic ischemic tissue, but also in normal colon tissue and inflammatory disorders.

Study of some Pulmonary Function Tests in Children with Sickle Cell Anemia: Correlation with Iron Overload

Background: Sickle cell disease is hereditary hemoglobinopathy characterized by abnormal hemoglobin production, hemolytic anemia, and intermittent occlusion of small vessels, leading to acute and chronic tissue ischemia, and organ damage. Pulmonary function tests can differentiate between obstructive or restrictive lung diseases, assess the severity of the disease and evaluate the degree of efficacy of therapy in patients with sickle cell anemia. Objective: The aim of this study was to study pulmonary function tests in children with sickle cell anemia and its correlation with iron overload. Patients and methods: This study was carried out on 40 children with sickle cell anemia under follow up at Hematology unit, Pediatric Department, Tanta university hospital in the period between October 2012 and August 2014 including 24 males and 16 females with their age ranging from 9– 15 years and mean age value of 12.7+2.2 years and 40 healthy children as a control group including 22 males and 18 females with their age ranging from 7 – 15 years and mean age value of 10.3+2.36 years. All patients were subjected to the following investigations: Complete blood count, HB electrophoresis, serum ferritin, serum iron, total iron binding capacity and pulmonary function tests. Results: There were significantly higher serum ferritin, serum iron and lower TIBC in patients than controls, significantly lower Forced vital capacity (FVC), Forced expiratory volume in one second (FEV1), FEV1/FVC ratio, Peak expiratory flow rate, and Forced expiratory Flow in patients compared with controls, significant negative correlation between serum ferritin and both of FVC and FEV1 in studied patients. There were restrictive spirometric pattern in 30 patients with sickle cell anemia (75%) and mixed obstructive and restrictive pattern in 10 patients (25%) with highly significant differences between patients and controls regarding restrictive and mixed pattern of spirometry. Conclusion: Most SCD patients show abnormal pulmonary functions, predominantly of restrictive pattern. Recommendations: Patients with SCD should undergo pulmonary function tests to detect and confirm lung disease for early and proper therapeutic intervention.

P76: H2S and Nitrite anion: Partners in preserving ischemic tissue function

Hydrogen sulfide (H2S) therapy has been reported to modulate vascular function during ischemia. Although nitric oxide synthase (NOS) dependent mechanisms of H2S mediated protection during tissue ischemia have been reported, little is known regarding the relationship between H2S and nitrite metabolism under similar conditions. We examined the NOS independent but nitrite dependent molecular mechanisms involved in H2S regulation of NO bioavailability and the importance of these effects during chronic tissue ischemia. Permanent unilateral hind limb ischemia was induced in wild type and eNOSKO mice by left femoral artery ligation and excision. Mice were assigned to four different groups (n = 8, each group) and treated with PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S, a H2S donor) twice daily by retro-orbital injection. Hind limb perfusion was determined using laser Doppler flowmetry. Angiogenic and cell proliferation index were detected by the ratio of CD31 to DAPI and Ki67/DAPI positive staining respectively. NO generation by Na2S and nitrite reduction in the presence of xanthine oxidase (XO) in endothelial cells was measured using a NO chemiluminescent analyzer. XO activity, cGMP, and VEGF expression were determined by ELISA. Blood perfusion, angiogenic index, cell proliferation index, and VEGF levels were all significantly increased in mice treated with Na2S compared to PBS control. These effects were blunted by cPTIO treatment both in vivo and in vitro suggesting the involvement of NO in H2S mediated ischemic tissue protection. Na2S and nitrite interaction in hypoxic endothelial cells increased NO production in a XO dependent manner. XO activity was increased in Na2S treated mice compared to PBS control. Both in vitro and in vivo data indicate the involvement of XO mediated nitrite reduction to NO that is dependent on H2S. Na2S therapy also increased cGMP levels, which were still observed in eNOS KO mice. Lastly, VEGF164 aptamer inhibited sulfide induced augmentation of blood flow in mice ischemic tissue indicating VEGF164 as a primary downstream target of H2S mediated nitrite reduction to NO under ischemic conditions. Na2S therapy restores ischemic tissue perfusion through a XO mediated nitrite reduction (NO/cGMP/VEGF) pathway. Thus, H2S mediated NO generation from nitrite would be a novel therapeutic option during ischemic vascular remodeling in the state of metabolic syndrome where NOS dependent pathways are defective. Nothing to disclose.

Post-Bypass Extensive Ascites due to Splanchnic Bypass and the Effectiveness of Hyperalimentation Treatment

Reperfusion edema may develop in the early periods of chronic ischemic tissue reperfusion. Reperfusion edema may be represented after the splanchnic bypass with ascites, abdominal distension, and liver and kidney function impairment. In this article, we are reporting the hyperalimentation treatment and its results for the common ascites and hepatorenal syndrome, after a coeliac and superior mesenteric artery bypass.

Heart Failure with preserved ejection fraction (HFPEF) and pulmonary hypertension may complicate cases of Sickle Cell Disease in Saudi patients originally from the Eastern Province, Saudi Arabia

Sickle cell disease is an important chronic inherited haemoglobin disorder, associated with recurrent vaso-occlusive and haemolytic crises and chronic tissue ischemia which may adversely affect any organ system including the cardiovascular system. Our objectives were to evaluate the cardiac performance in general with concentration on LV diastolic function and pulmonary artery pressure in young adult and adult Saudi patients originally from the Eastern province of Saudi Arabia. Forty-five patients with sickle disease were recruited for echocardiographic study while 45 controls, matched for age and sex, served as controls. Left and right ventricular dimensions and wall thicknesses of the LV, LV mass, LV mass index and LV contractility variables were obtained. Left atrial dimension and volume and pulmonary artery systolic pressure were also estimated. We evaluated also parameters of LV diastolic function including early and late atrio-ventricular flow velocities (E and A wave, respectively), E/A ratio, deceleration time (DT), A wave duration (A Dur.), and LV isovolumic relaxation time (IVRT). Other parameters of LV diastolic function including evaluation of Tissue Doppler Velocities, such as lateral annular e′ wave, a′ wave, e′/a′ ratio and E/e′ ratio were evaluated. There were increases in the LV dimensions, LV volumes, stroke volume, and LV mass indexes of the SCD patients. The preload was increased (LV diastolic volume) and afterload was decreased (low diastolic blood pressure). The LV ejection fraction was equivalent. However, there were evidences of LV diastolic dysfunction in 24%, and PH in 40% of the SCD patients. The mean left atrial volume was also increased in the SCD patients. Left ventricular diastolic dysfunction (Heart Failure with Preserved Ejection Fraction – HFPEF) and pulmonary hypertension may complicate cases of the Arab-Indian haplotype of sickle cell disease.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia‐Inducible Factor‐1α and Vascular Endothelial Growth Factor–Dependent Angiogenesis

BackgroundHydrogen sulfide (H2S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H2S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H2S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses.Methods and ResultsThe unilateral, permanent femoral artery ligation model of hind‐limb ischemia was performed in C57BL/6J wild‐type and endothelial NO synthase–knockout mice to evaluate exogenous H2S effects on NO bioavailability and ischemic revascularization. We found that H2S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H2S increased ischemic tissue xanthine oxidase activity, hind‐limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H2S treatment increased ischemic tissue and endothelial cell hypoxia‐inducible factor‐1α expression and activity and vascular endothelial growth factor protein expression and function in a NO‐dependent manner that was required for ischemic vascular remodeling.ConclusionsThese data demonstrate that H2S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling.

Beets, Bacteria, and Blood Flow

Angiogenesis and its regulation is a crucial physiological response to chronic tissue ischemia causing increased microvessel growth along with collateral arteriolar remodeling to restore blood flow to deprived tissues.1 Vascular occlusion, as seen in coronary and peripheral artery disease, often progresses to chronic tissue ischemia without effective vascular remodeling responses.2 Thus, therapeutic revascularization has an important role to play in the management of patients with coronary or peripheral artery disease to facilitate ischemic tissue reperfusion and limit cellular injury and loss.3 Unfortunately, successful realization of therapeutic angiogenesis remains elusive despite numerous experimental and clinical studies. Article see p 1983 Nitric oxide (NO) regulates several important vascular responses in chronic tissue ischemia, including angiogenesis, vasodilation, anti-inflammation, and apoptosis, to name a few.4,5 Potentiation of NO augments therapeutic angiogenesis6; however, the activity of nitric oxide synthase enzymes and its cofactors are compromised during ischemia.7 Despite that fact, several studies indicate that NO metabolites nitrite (NO 2 − ) and nitrate (NO 3 − ), once considered to be inert by-products of NO, can be chemically reduced back to NO in ischemic tissues through multiple reduction mechanisms.8 In this way, the nitrate/nitrite/NO pathway may serve as an alternative (perhaps …

P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway

Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine γ-lyase (CSE), cystathionine β- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na 2 S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe. SPY imaging was performed to determine the collateral formation in different time points. Capillary myofiber ratio was determined by NOVA Red staining with CD31 antibody. Mature vessel density and proliferation index were determined by the ratio of smooth muscle actin (SMA) to DAPI and Ki67 to DAPI positive areas respectively. TUNEL assay was measured to investigate the apoptosis. Nitric oxide (NO) was measured by NO analyzer (NOA). CPTIO (1 mg/kg/day) was injected intra-peritoneally to determine the effect of NO in sulfide induced ischemic angiogenesis. VEGF aptamer (50 mg/kg/day) was injected locally to the ischemic muscle to determine the role of VEGF in sulfide induced ischemic angiogenesis. Lastly, VEGF expression was determined by ELISA. Blood perfusion, blush rate by SPY imaging, angiogenic index, proliferation index and mature vessel density were significantly decreased in CSEKO mice compared WT mice indicating the role of sulfide on ischemic angiogeneis and arteriogenesis. Na 2 S dose dependently increases the angiogenesis index, proliferation index, mature vessel density and restores the blood flow in WT mice. Hydrogen sulfide also rescues the impaired blood flow in CSEKO mice by increasing angiogenic index, proliferation index and mature vessel density. Blood tissue perfusion, proliferation index, capillary to myofiber ratio and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limbs of aged diabetic mice treated with Na 2 S compared to PBS control. There was no effect of sulfide on blood glucose, body weight and lipid profiles of diabetic mice.Total NO production was increased in sulfide treated group compared to PBS which was inhibited by CPTIO treatment. Lastly, VEGF164 aptamer blocked sulfide induced augmentation of blood flow and reduced the expression of VEGF164 in WT and diabetic mice hind limb ischemia indicating VEGF164 as the sulfide induced angiogenic stimulator. Sodium sulfide therapy restores tissue perfusion of critical limb ischemia by increasing production of NO, increasing expression of VEGF and preventing apoptosis in diabetic mouse chronic hind limb ischemia which may be beneficial for the diabetic patient with peripheral vascular disease.

Echocardiographic evaluation of left ventricular diastolic and systolic function in Saudi patients with sickle cell disease

Background and objectivesSickle cell disease (SCD) is a chronic, inherited haemoglobin disorder, associated with recurrent vaso-occlusive and haemolytic crises and chronic tissue ischemia which may adversely affect any organ system. Our objectives were to evaluate the left ventricular (LV) systolic and diastolic functions in Saudi patients with SCD originally from the Eastern Province of Saudi Arabia. Design and settingProspective hospital based echocardiography study on adolescent and adult patients with SCD. MethodsForty-five patients with SCD were recruited for echocardiographic study while 45 patients, matched for age and sex, served as controls. Left and right ventricular dimensions and LV wall thicknesses, LV mass index (LVMI) and LV contractility variables were obtained. Left atrial dimension and volume and pulmonary artery systolic pressure (PASP) were also estimated. We also evaluated parameters of LV diastolic function, including early and late mitral flow velocities (E and A wave respectively), E/A ratio, deceleration time (MVDT), A wave duration (MVA D), LV isovolumic relaxation time (IVRT), and tissue Doppler velocities, such as lateral annular e‘ wave, a‘ wave, e‘/a‘ ratio and E/e‘ ratio. ResultsThere were increases in the LV dimensions, LV volumes, stroke volume, and LVMI of the SCD patients. The preload was increased (LV diastolic volume) and afterload was decreased (low diastolic blood pressure). The LVEF was equivalent, though there was evidence of LV diastolic dysfunction in 24%, and pulmonary hypertension (PH) in 40% of the SCD patients. The mean left atrial volume (LAV) was also increased in the SCD patients. ConclusionLV diastolic dysfunction (heart failure with preserved ejection fraction) and PH may complicate cases of the Arab-Indian haplotype of SCD.

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.

Abstract 272: Impaired Arteriogenesis in Cystathionine Gamma-Lyase (CSE) and Endothelial Nitric Oxide Synthase (eNOS) Mutant Mice

Introduction: Arteriogenesis is an important process involving remodeling of collateral vasculature during chronic or intermittent tissue ischemia. CSE and eNOS are the two principle enzymes for hydrogen sulfide (H 2 S) and Nitric Oxide (NO) generation. Although role of NO during arteriogenesis is partially known, the role of CSE dependent H 2 S production during arteriogenesis remains unknown. Hypothesis/objective: We sought to determine the role of CSE generated H 2 S on arteriogenesis activity of CSE knockout (CSEKO) mice compared to eNOS knock out (eNOSKO) and wild type (WT) mice during hind limb ischemia. Method: Permanent unilateral hind limb ischemia was induced in 12-week-old CSE KO, eNOS KO, and WT mice and studied until day 21. Tissue perfusion was measured using indicator dye blush rates with the SPY Imager system over time along with laser doppler flowmetry. Temporal development of arterial remodeling was also observed using SPY imaging, and vessel number, length and branch morphometry determined over time. Microfil vascular casting was performed to further examine the number of collaterals in ischemic muscles at the end of the study. Histological arteriogenesis was evaluated by vascular smooth muscle actin (SMA) positive vessel density with dual fluorescence staining of anti-CD31 (endothelial positive cell) and anti-α-SMA antibodies. Results: Tissue perfusion was significantly impaired in CSE KO and eNOS KO mice compared to WT mice at different time points throughout the study period. Both CSE KO and eNOS KO mice showed significantly less blush rate than WT mice. SMA positive vessel density was also significantly less in CSE KO and eNOS KO mice than that in WT mice. Conclusion: CSE generated H 2 S and eNOS derived NO play important roles in regulating ischemic arterial remodeling as demonstrated in our studies. The mechanism of impaired arteriogenesis in both mutant mice and the interaction between these two gasotransmitters has yet to be elucidated.

Nitrite and nitric oxide metabolism in peripheral artery disease

Peripheral artery disease (PAD) represents a burgeoning form of cardiovascular disease associated with significant clinical morbidity and increased 5year cardiovascular disease mortality. It is characterized by impaired blood flow to the lower extremities, claudication pain and severe exercise intolerance. Pathophysiological factors contributing to PAD include atherosclerosis, endothelial cell dysfunction, and defective nitric oxide metabolite physiology and biochemistry that collectively lead to intermittent or chronic tissue ischemia. Recent work from our laboratories is revealing that nitrite/nitrate anion and nitric oxide metabolism plays an important role in modulating functional and pathophysiological responses during this disease. In this review, we discuss experimental and clinical findings demonstrating that nitrite anion acts to ameliorate numerous pathophysiological events associated with PAD and chronic tissue ischemia. We also highlight future directions for this promising line of therapy.

Overexpression of VEGF 165 b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, fibrosis, and autoimmunity that affects the skin and internal organs. Although in SSc there is a lack of sufficient angiogenic response to chronic tissue ischemia culminating in the loss of capillary vessels, the expression of vascular endothelial growth factor-A (VEGF) has paradoxically been shown to be upregulated in SSc skin and circulation. However, previous studies in the field did not distinguish between the proangiogenic VEGF 165 and antiangiogenic VEGF 165 b isoforms that are generated by alternative splicing in the terminal exon of VEGF pre-RNA. In the present study, we investigated whether VEGF isoform expression could be altered in skin and circulation of SSc patients. Using RT-PCR, quantitative real-time PCR, Western blotting, immunohistochemistry and confocal microscopy, we could show that the VEGF 165 b splice variant was selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF 165 b expression correlated with increased expression of profibrotic transforming growth factor-β1 (TGF-β1) and serine/arginine protein 55 (SRp55) splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. ELISA on plasma samples revealed that circulating levels of VEGF 165 b were significantly higher in SSc patients than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF 165 b than healthy MVECs (H-MVECs). TGF-β1 upregulated the expression of VEGF 165 b and SRp55 in both SSc- and H-MVECs. In SSc-MVECs, VEGF receptor-2 (VEGFR-2) was overexpressed, but its phosphorylation and ERK1/2 downstream signaling were impaired. Recombinant human VEGF 165 b and SSc-MVEC–conditioned medium inhibited VEGF 165 -mediated VEGFR-2 phosphorylation, ERK1/2 activation and capillary morphogenesis on Matrigel in H-MVECs. The addition of anti-VEGF 165 b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc-MVECs and could be ameliorated by treatment with recombinant VEGF 165 and anti-VEGF 165 b blocking antibodies. In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia. The combination of proangiogenic VEGF 165 administration and VEGF 165 b neutralization might represent a potential therapeutic strategy to promote effective angiogenesis and capillary regeneration in SSc.

Dipyridamole reverses peripheral ischemia and induces angiogenesis in the Db/Db diabetic mouse hind-limb model by decreasing oxidative stress

Dipyridamole anti-platelet therapy has previously been suggested to ameliorate chronic tissue ischemia in healthy animals. However, it is not known if dipyridamole therapy represents a viable approach to alleviating chronic peripheral tissue ischemia associated with type 2 diabetes. Here we examine the hypothesis that dipyridamole treatment restores reperfusion of chronic hind-limb ischemia in the murine B6.BKS-Lepr db/db diabetic model. Dipyridamole therapy quickly rectified ischemic hind-limb blood flow to near preligation levels within 3 days of the start of therapy. Restoration of ischemic tissue blood flow was associated with increased vascular density and endothelial cell proliferation observed only in ischemic limbs. Dipyridamole significantly increased total nitric oxide metabolite levels in tissue, which were not associated with changes in endothelial NO synthase expression or phosphorylation. Interestingly, dipyridamole therapy significantly decreased ischemic tissue superoxide and protein carbonyl levels, identifying a dominant antioxidant mechanistic response. Dipyridamole therapy also moderately reduced diabetic hyperglycemia and attenuated development of dyslipidemia over time. Together, these data reveal that dipyridamole therapy is an effective modality for the treatment of chronic tissue ischemia during diabetes and highlights the importance of dipyridamole antioxidant activity in restoring tissue NO bioavailability during diabetes.