Setting Of Chronic Ischemia Research Articles

Abstract 3024: Mitochondrial Volume Abnormalities In Gastrocnemius Muscle Of Patients With Peripheral Artery Disease

Background: The mechanisms of claudication in patients with peripheral artery disease (PAD) are poorly understood. Mitochondria are responsible for ATP production during aerobic conditions but may become structurally impaired in patients with chronic ischemia and PAD. Research Question: Are mitochondrial volumes different in patients with PAD compared with age-matched controls? Aims: To better understand mitochondrial abnormalities and ultrastructure in patients with PAD. Methods/Approach: Patients with PAD (defined as ABI <0.85) and age-matched controls (ABI 1.00-1.40) were recruited and underwent gastrocnemius muscle biopsy. Muscle samples were collected in optimal cutting temperature compound. Mitochondria were labeled with cytochrome oxidase IV antibody and Cy3 fluorescence secondary antibody and images were rendered in three dimensions using structured illumination microscopy to quantify mitochondrial size, content, and morphology. Comparisons were done with a Mann-Whitney U test. Results/Data: Forty-eight subjects (median [IQR] age 66.5 [59.0, 74.0] years, 32 (66.7%) male) were recruited and underwent gastrocnemius muscle biopsy, including 16 controls and 32 with PAD. The largest singular mitochondrial volume was greater in those with PAD (22.6 vs. 4.2μm 3 , p=0.01, Figure 1A) but the median mitochondrial volume was smaller (0.07 vs. 0.01μm 3 , p=0.03, Figure 1B). While no control mitochondria were larger than 7.2μm 3 , 1% of mitochondria in patients with PAD were larger than 10μm 3 (Figure 1C). There was no difference in the total mitochondrial volume or content between PAD and controls. Conclusion: Patients with PAD have larger maximum but smaller median mitochondria sizes in the gastrocnemius muscle, suggesting greater heterogeneity of mitochondrial size in those with PAD. The changes in mitochondrial morphology may result in mitochondrial inefficiency and an impaired compensatory response in the setting of chronic ischemia from PAD.

Abstract P3111: Sex Differences In The Proteomic Response To Chronic Myocardial Ischemia In Swine

Intro: There are known sex differences in response to ischemic insult yet the mechanisms behind these differences have not been fully elucidated. Previous work in our lab has demonstrated female sex confers a relative protective effect on CO in the setting of chronic ischemia. The aim of this study was to use total proteomic analysis in order to uncover the mechanisms behind sex specific responses to myocardial ischemia in a porcine model. Methods: Eight 11-week old pigs (3 female, 5 male) underwent surgical placement of an ameroid constrictor to the left circumflex artery via left thoracotomy in order to induce chronic myocardial ischemia. After seven weeks, pigs were euthanized and myocardial tissue was harvested for total proteomic analysis. Results: When compared with non-ischemic male tissue non-ischemic female tissue demonstrated upregulation of two variants of LMOD2 and down regulation of five variants of ACADS (Figure 1A). However, when comparing ischemic female tissue with ischemic male tissue there were 626 upregulated proteins and 194 down regulated proteins (Figure 1B). Enrichment analysis revealed upregulation of certain metabolic pathways including the citric acid cycle and glycolysis/gluconeogenesis (Figure 1C) while there was down regulation in pathways involved in cardiac muscle contraction and dilated cardiomyopathy (Figure 1D). There was no difference in serum glucose, HDL, LDL, or total cholesterol (P > 0.05 for all) (Figure 1E). Conclusion: There were clear sex differences in response to chronic cardiac ischemia. Proteins involved in metabolic pathways and myocyte function appear to be particularly affected.

Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery.

MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p-transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1 /Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

S2-1 Hydrogen sulfide in heart failure

Background Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro model systems and to promote vessel growth in the setting of chronic ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donors, SG-1002 and diallyl trisulfide (DATS), in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis. Studies were also performed to determine the mechanisms by which H2S protects the heart during heart failure. Methods and results Transverse aortic constriction was performed in mice (C57BL/6J; 8–10 weeks of age). Mice received either vehicle or diallyl trisulfide (200 μg/kg) or SG-1002 (20 mg/kg) starting 24 h after transverse aortic constriction and were followed for up to 12 weeks using echocardiography. H2S therapy with H2S donors significantly improved left ventricular remodeling and preserved left ventricular function in the setting of transverse aortic constriction. H2S therapy increased the expression of the proangiogenic factor, vascular endothelial cell growth factor, and decreased the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial NO synthase and the bioavailability of NO. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts. In additional studies we determined that H2S therapy resulted in the activation of endothelial nitric oxide synthase (eNOS) via phosphorylation of serine1177 and increased nitric oxide (NO) bioavailability. Interestingly, we determined that eNOS was uncoupled in CSE KO mice with reduced levels of H2S and NO coupled with an exacerbated injury response to acute myocardial infarction. Conclusions These results suggest that H2S therapy attenuates left ventricular remodeling and dysfunction in the setting of heart failure by creating a proangiogenic environment for the growth of new vessels. A major mechanism related to these cytoprotective actions is the activation of eNOS and increased nitric oxide bioavailability.

Ethanol Promotes Arteriogenesis and Restores Perfusion to Chronically Ischemic Myocardium

Moderate alcohol consumption is known to be cardioprotective compared with either heavy drinking or complete abstinence. We assessed the hypothesis that ethanol supplementation would improve myocardial function in the setting of chronic ischemia. Sixteen male Yorkshire swine underwent placement of an ameroid constrictor into the left circumflex artery to induce chronic myocardial ischemia. Postoperatively, animals were supplemented with either 90 mL of ethanol (EtOH) daily (50%/V, EtOH) or 80 g of sucrose of equal caloric value (SUC), serving as controls. Seven weeks after ameroid placement, arteriolar density (1.74 ± 0.210% versus 3.11 ± 0.368% area of arterioles per low-powered field in sucrose (SUC) versus EtOH; P=0.004), myocardial perfusion (ratio of blood flow to the at-risk myocardium compared with the normal ventricle during demand pacing was 0.585 ± 0.107 versus 1.08 ± 0.138 for SUC versus EtOH; P=0.014), and microvascular reactivity were significantly increased in ethanol-treated animals compared with controls in the at-risk myocardium. Analysis of vascular endothelial growth factor and NOTCH pathway signaling suggested proneovascular and proliferative activity in the ischemic area. The average peak blood alcohol level in the treatment group was 40 ± 4 mg/dL, consistent with levels of moderate drinking in humans. Ethanol supplementation increased arteriolar density and significantly improved myocardial perfusion and endothelium-dependent vasorelaxation in chronically ischemic myocardium. These findings suggest that, at moderate doses, ethanol directly promotes vasculogenesis and improves microvascular function, resulting in significant improvements in myocardial perfusion in the setting of chronic ischemia.

Abstract 19114: Ultrasound-Targeted Delivery of microRNA-126 for Therapeutic Angiogenesis

Introduction: MicroRNA-126 (miR-126) is an endothelial-specific microRNA that regulates angiogenesis by blocking endogenous inhibitors of vascular endothelial growth factor (VEGF), Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K). We hypothesized that ultrasound-mediated delivery of miR-126 would result in improved angiogenesis in the setting of chronic ischemia. Methods: Naked mature miR-126 was synthesized for transfection of rat specific miR-126 (22bp). Unilateral hindlimb ischemia was created by left femoral artery ligation in F-344 rats (n=45). At day 14 post-ligation, microvascular blood flow (MBF) in the distal hindlimb muscles were assessed by contrast-enhanced ultrasound (CEU). Ultrasound-mediated gene delivery (UMGD) of miR-126 (1x109 cationic microbubbles + 2 μ g of miR-126) (n=12) or scrambled miR (miR-SCR) (n=12) was performed, with control animals (n=11) receiving no treatment. An additional group (n=10) received UMGD of miR-126 (2 ug) three times every 48hours (day14, day 16 and day18). CEU perfusion was re-assessed at day 28, and transfection was assessed by real time PCR. Results: At day 14, prior to miR-126 delivery, normalized MBF for the ischemic muscle was similarly reduced in all treatment groups (55-60% of normal). At day 28, MBF remained unchanged in control and miR-SCR treated groups (0.61±0.08 vs 0.62±0.09, and 0.59±0.06 vs 0.63±0.07, respectively. In miR-126 treated animals, MBF improved (0.59±0.07 vs 0.78±0.06, p < 0.001 versus control and miR-SCM). After triple delivery of miR-126, further improvement in perfusion was noted (0.58±0.04 vs 0.88±0.03, p < 0.001 versus control and miR-SCM, and p <0.01 versus single miR-126). Real time PCR showed a 14.3±3.4 fold increase over control at 3h post transfection of miR-126 that persisted to day 3 post-UMGD, and normalized by day7 (n=3 per time point). Conclusions: UMGD of miR-126 results in improved perfusion in the setting of chronic hindlimb ischemia. Multi-gene delivery of miR-126 showed an incremental angiogenic response as compared to single gene delivery.

Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.

Loss of Hypoxia-Inducible Factor Prolyl Hydroxylase Activity in Cardiomyocytes Phenocopies Ischemic Cardiomyopathy

Ischemic cardiomyopathy is the major cause of heart failure and a significant cause of morbidity and mortality. The degree of left ventricular dysfunction in this setting is often out of proportion to the amount of overtly infarcted tissue, and how decreased delivery of oxygen and nutrients leads to impaired contractility remains incompletely understood. The Prolyl Hydroxylase Domain-Containing Protein (PHD) prolyl hydroxylases are oxygen-sensitive enzymes that transduce changes in oxygen availability into changes in the stability of the hypoxia-inducible factor transcription factor, a master regulator of genes that promote survival in a low-oxygen environment. We found that cardiac-specific PHD inactivation causes ultrastructural, histological, and functional changes reminiscent of ischemic cardiomyopathy over time. Moreover, long-term expression of a stabilized hypoxia-inducible factor alpha variant in cardiomyocytes also led to dilated cardiomyopathy. Sustained loss of PHD activity and subsequent hypoxia-inducible factor activation, as would occur in the setting of chronic ischemia, are sufficient to account for many of the changes in the hearts of individuals with chronic coronary artery disease.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1beta (3.8X) and TNF-alpha (6.0X). IFN-gamma was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Segmentation-based algorithms to quantify nonviable myocardium after delayed contrast-enhanced computed tomography: defining what’s dead

The ability to distinguish dysfunctional but viable myocardium from nonviable tissue after acute or chronic ischemia has important implications for the therapeutic management of patients with coronary artery disease. In the acute phase, myocardial revascularization in patients with viable myocardium can improve ventricular dysfunction 1 Alderman E.L. Fisher L.D. Litwin P. Kaiser G.C. Myers W.O. Maynard C. Levine F. Schloss M. Results of coronary artery surgery in patients with poor left ventricular function (CASS). Circulation. 1983; 68: 785-795 Crossref PubMed Scopus (698) Google Scholar and long-term survival, 2 Pagley P.R. Beller G.A. Watson D.D. Gimple L.W. Ragosta M. Improved outcome after coronary bypass surgery in patients with ischemic cardiomyopathy and residual myocardial viability. Circulation. 1997; 96: 793-800 Crossref PubMed Scopus (358) Google Scholar whereas image-based characterization of myocardial scar structure in the setting of chronic ischemia can identify those patients with hibernating myocardium who may achieve functional systolic recovery with revascularization. 3 Wakimoto H. Maguire C.T. Kovoor P. Hammer P.E. Gehrmann J. Triedman J.K. Berul C.I. Induction of atrial tachycardia and fibrillation in the mouse heart. Cardiovasc Res. 2001; 50: 463-473 Crossref PubMed Scopus (78) Google Scholar The assessment of both acute and chronic myocardial viability using delayed enhanced magnetic resonance imaging (de-MRI) is considered the standard for myocardial viability assessment. As the clinical indications for implantable cardiac defibrillators and biventricular pacing therapy continue to expand, however, development and validation of alternative imaging methods with similar anatomic, functional, and viability imaging capabilities are needed to accommodate this growing population of patients.

Coronary microvascular dysfunction in the setting of chronic ischemia is independent of arginase activity

Background Chronic myocardial ischemia induces endothelial dysfunction in the coronary microcirculation resulting in impaired nitric oxide signaling. This dysfunction has wide-ranging effects including impaired tissue perfusion and is implicated in impairment of the angiogenic process in settings of endothelial dysfunction. We hypothesized chronic myocardial ischemia results in increased activity of Arginase I, diminishing bioavailability of l-arginine, the substrate for endothelial nitric oxide production. Methods Chronic myocardial ischemia was induced for 7-weeks in 6 Yucatan miniswine utilizing an ameroid constrictor placed around the left circumflex coronary artery. Ischemic and non-ischemic tissue was harvested at the 7-week time point. Expression of Arginase I, eNOS, and phospho-eNOS was assessed utilizing Western blotting. Arginase activity was measured. Immunofluorescent staining assessed expression of Arginase I between ischemic and non-ischemic microvessels. Coronary microvascular relaxation studies were performed. Results Arginase I expression, activity, and staining was similar between ischemic and non-ischemic territories. Significant impairments in coronary microvascular relaxation were observed in microvessels from the ischemic territory in response to endothelial-dependent agents but remained similar between territories in response to endothelial independent agents. Regression analysis between arginase activity and degree of microvascular vasorelaxation demonstrated no significant correlation. Conclusions Coronary microvascular dysfunction in the setting of chronic myocardial ischemia occurs independently of Arginase I activity and expression. Alternative therapeutic strategies focusing away from arginine may be need for the treatment of this dysfunction.

Sildenafil Promotes Ischemia-Induced Angiogenesis Through a PKG-Dependent Pathway

Peripheral artery disease (PAD) is a prevalent cardiovascular disorder that results in tissue ischemia which can progress to critical limb ischemia. Restoration of tissue perfusion in the setting of chronic ischemia through stimulation of arteriogenesis and angiogenesis remains a key therapeutic target for PAD. However, experimental therapeutics, including growth factor and gene therapy, have had little clinical success indicating the need for a better understanding of molecular pathways required for therapeutic angiogenesis. Here we report that phosphodiesterase-5 inhibition by sildenafil significantly increases vascular perfusion, tissue blood flow, and vascular density during chronic ischemia of the mouse hind limb. Importantly, sildenafil therapy did not alter any of these parameters in nonischemic limbs. Sildenafil increased tissue cGMP levels independently of increases in nitric oxide production, and sildenafil therapy stimulated angiogenesis in ischemic limbs of eNOS-/- and iNOS-/- mice. Lastly, sildenafil-mediated angiogenic activity was blocked by inhibition of protein kinase G using the PKG antagonist DT-3. These data demonstrate that sildenafil therapy results in increased angiogenic activity through a PKG-dependent pathway that is independent of nitric oxide production or NOS activity and identify the angiogenic therapeutic potential of sildenafil for critical limb ischemia.