Priming with ligands secreted by human stromal progenitor cells promotes grafts of cardiac stem/progenitor cells after myocardial infarction.

Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells including fibroblasts and stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/progenitor cells in culture. We hypothesized that secreted factors in conditioned medium (CdM) from adult bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/CPCs) and improve graft success after myocardial infarction (MI). Incubation of adult rat CPCs in CdM from human MSCs isolated by plastic adherence or by magnetic sorting against CD271 (a.k.a., p75 low-affinity nerve growth factor receptor; p75MSCs) induced phosphorylation of STAT3 and Akt in CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30× p75MSC CdM for 30 minutes prior to transplantation into subepicardial tissue 1 day after MI markedly increased engraftment compared with vehicle priming. Screening CdM with neutralizing/blocking antibodies identified connective tissue growth factor (CTGF) and Insulin as key factors in p75MSC CdM that protected CPCs. Human CTGF peptide (CTGF-D4) and Insulin synergistically promoted CPC survival during hypoxia in culture. Similar to CdM priming, priming of CSCs with CTGF-D4 and Insulin for 30 minutes prior to transplantation promoted robust engraftment, survival, and migration of CSC derivatives at 1 week and 1 month after MI. Our results indicate that short-term priming of human CSCs with CTGF-D4 and Insulin may improve graft success and cardiac regeneration in patients with MI.

The efficacy and tolerability of azilsartan in mice with left ventricular pressure overload or acute myocardial infarction.

Angiotensin II receptor blockers (ARBs) are used for the treatment of patients with heart failure and hypertension. Yet their safety has been questioned by some who observed delayed cardiac healing and scar thinning after myocardial infarction (MI). To clarify potential efficacy and safety of ARBs, we administered Azilsartan medoxomil, a prodrug of an ARB (Takeda Pharmaceutical Company Limited), assessed cardiac fibrosis (hydroxyproline content), left ventricular (LV) wall thickness (premortem echocardiography and caliper measurement at necropsy), and LV mass and cardiac function with high-resolution ultrasound in mice with either surgically induced LV pressure overload (aortic banding) or acute MI. Drug-treated aortic-banded mice exhibited less LV wall thickness, hypertrophy, and dilation compared with that exhibited by controls. Survival in drug-treated MI mice was greater though not significantly. Drug-treated mice with acute MI exhibited less cardiomyocyte injury reflected by LV creatine kinase content and less LV hypertrophy and dilation. Thus, Azilsartan exerted favorable biological effects on the hearts of mice subjected to LV pressure overload or MI without compromising survival consistent with its potential utility and tolerability in patients with analogous conditions.

The angiotensin receptor blocker, azilsartan medoxomil (TAK-491), suppresses vascular wall expression of plasminogen activator inhibitor type-I protein potentially facilitating the stabilization of atherosclerotic plaques.

Increased expression of plasminogen activator inhibitor type-I (PAI-1) in vessel walls seems to accelerate atherosclerosis. Angiotensin II can increase the synthesis of PAI-1. Inhibition of this process may facilitate migration of vascular smooth muscle cells (VSMCs) stabilizing atherosclerotic plaques. To determine whether the inhibition of the angiotensin II type 1 receptor can blunt the expression of PAI-1 protein in the aortic wall, we administered azilsartan medoxomil (AZL-M), a prodrug of an angiotensin II type 1 receptor blocker developed by the Takeda Pharmaceutical Company Limited, for 16 weeks to ApoE knockout mice on a high fat diet rendered overexpressors of PAI-1 in VSMCs. Homogenates of the pooled aortas from each group were assayed for PAI-1 by enzyme-linked immunosorbent assay. Cellularity of atherosclerotic lesions was assessed by 4',6-diamidino-2-phenylindole staining in sections of aortic lesions, and collagen content in the lesions was quantified by immunohistochemistry. Aortic wall PAI-1 was decreased by each of the 3 dosage regimens of AZL-M (0.1-10 mg/kg). Cellularity and collagen were increased in lesions from mice given AZL-M, consistent with the development of more stable plaques. Accordingly, the suppression of PAI-1 expression by AZL-M may attenuate the evolution of atherosclerotic plaques vulnerable to rupture.

Vascular rhexis in mice subjected to non-sustained myocardial ischemia and its therapeutic implications.

We previously described the death of vascular cells (vascular rhexis) following persistent coronary occlusion. The present study was designed to determine whether non-sustained ischemia can initiate vascular rhexis and if so, whether relatively brief ischemic insults are sufficient. C57BL6 mice were subjected to coronary ligation for 15 min or 3 h followed by reperfusion. Soluble fractions of left ventricular (LV) homogenates were obtained 48 h after the onset of transitory coronary occlusion. They were assayed by Western blotting for quantification of alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) that we have shown reflect vascular rhexis delineated immunohistochemically. Non-sustained coronary occlusion for 3 h initiated vascular rhexis evident 45 h after reperfusion, but not earlier, as judged from Western blotting of α-SMA and SM-MHC. The number of small- and medium-sized vessels in the previously ischemic zones was reduced at 45 h after reperfusion as well. Thus, vascular rhexis occurs after ischemia as brief as 3 h but evolves slowly and is not evident for 45 h. The delayed disintegration of the vasculature makes it likely that it can be ameliorated by interventions initiated after non-sustained ischemia, rendering it an attractive target for diminution of phenomena such as late negative LV remodeling, and 'no reflow.'

Failure of erythropoietin to render jeopardized ischemic myocardium amenable to incremental salvage by early reperfusion.

OBJECTIVES: Erythropoietin (EPO) has been thought to be capable of potentiating protection of jeopardized myocardium by reperfusion in evolving myocardial infarction. However, diversity in study design and measurements of infarct size in studies evaluating EPO has led to inconsistent results. We sought to characterize the effect of EPO on infarct size after myocardial ischemia and reperfusion with the use of assessment of left-ventricular (LV) creatine kinase (CK) depletion and echocardiography. METHODS: Acute coronary occlusion was induced in 10-week-old C57BL6 mice by left anterior descending coronary artery ligation for 3 h followed by 72 h of reperfusion. EPO (10,000 U/kg) or an equivalent amount of saline vehicle alone was injected intraperitoneally before ligation or immediately after the onset of reperfusion. Assays of residual LV CK activity and calculation of LV CK depletion were performed on LV homogenates harvested 72 h after onset of reperfusion for measurement of infarct size, and echocardiography was performed immediately before harvest of tissue for measurement of function. RESULTS: Mice administered EPO before ligation had similar infarct size (37.1+/-4.1%) and echo scores (22.9+/-0.4) compared with those in corresponding control mice administered saline (35.29+/-1.9 and 21.3+/-1.1%, respectively). Mice administered EPO after reperfusion had similar infarct size (39.1+/-4.8%) and echo scores (19.5+/-1.0) compared with those in corresponding control mice administered saline (40.3+/-4.9 and 21.5+/-1.9%, respectively). CONCLUSION: EPO does not protect ischemic myocardium such that reperfusion after 3 h can yield additional salvage.

A profibrotic effect of plasminogen activator inhibitor type-1 (PAI-1) in the heart.

Increased expression of PAI-1 is profibrotic in several organs. However, its potentially profibrotic effects in the heart subjected to infarction have not been elucidated. Accordingly, we induced coronary occlusion in 10-week-old mice congenic on a C57BL6 background and in mice overexpressing PAI-1 (PTG) in multiple tissues. Compared with C57BL6 control mice without myocardial infarction (MI), PTG mice exhibited consistently elevated PAI-1 in plasma at 16 weeks of age but virtually identical PAI-1 content in left ventricular (LV) myocardium. However, they exhibited a 2-fold increase in LV PAI-1 content 6 weeks after induction of MI (4.21 +/- 1.0 ng/ml tissue protein) compared with that in C57BL6 mice (2.04 +/- 0.5, P < 0.05). In 16-week-old mice, ultrasonically delineated LV fractional shortening (FS) was comparable in normal PTG and normal C57BL6 controls. However, 6 weeks after MI, PTG (n = 21) compared with C57BL6 (n = 14) mice exhibited markedly thinner LV posterior walls in both diastole (C57BL6 0.79 +/- 0.05 mm, PTG 0.55 +/- 0.06, P < 0.05) and systole (0.97 +/- 0.05 mm, 0.75 +/- 0.06, P < 0.05); increased end systolic LV dimensions (4.54 +/- 0.2 mm, 5.17 +/- 0.2, P < 0.05); and significantly depressed FS, more impaired LV segmental function, and greater mitral E wave amplitude. Compared with fibrosis assessed by Masson staining of sections from apex to base in C57BL6 mice (10.85 +/- 0.43% LV area), PTG mice exhibited 33% more LV fibrosis after MI (P < 0.05). Thus, PAI-1 is profibrotic in the heart subjected to infarction. Accordingly, overexpression of PAI-1 is a promising target for attenuation of heart failure after MI that may be exacerbated by fibrosis.

The magnitude and temporal dependence of apoptosis early after myocardial ischemia with or without reperfusion.

In view of the conventional wisdom in the cardiology literature that apoptosis is extensive early after myocardial ischemia, predicated largely from results with the TUNEL assay known to be nonspecific, this study was performed to delineate its extent with multiple assays and at multiple intervals. Coronary occlusion with and without subsequent revascularization was induced in 10-wk-old C57BL6 mice subjected to 1 or 4 h of transient ligation followed by 24 h of reperfusion, or 24 h persistent ligation. Apoptosis was quantified throughout the left ventricle immunohistochemically by assay of TUNEL, single-stranded DNA (ssDNA), and cleaved caspase 3; electron microscopy (EM); and activity assays of caspase 3 and 8. TUNEL staining was marked, but ssDNA and cleaved caspase 3 staining were significantly less (P<0.001 compared with TUNEL), and apoptosis defined by EM was virtually absent in all groups. Caspase 3 and caspase 8 activities per milligram protein were not significantly different from those in normal hearts. Only rare, potentially apoptotic cells were seen by EM in hearts from any group. Thus, the results with TUNEL were not specific, and the extent of apoptosis was markedly less than that predicated on the results with the TUNEL procedure. Apoptosis is de minimus early after transitory or persistent ischemia, though it is overestimated by TUNEL assays. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of myocardium early after ischemic insults.

A novel dual staining method for identification of apoptotic cells reveals a modest apoptotic response in infarcted mouse myocardium.

Confocal scanning laser microscopy was used to investigate the myocardium of control C57BL/6 and plasminogen activator inhibitor 1 knockout (PAI-1KO) mice 3 days following persistent ligation of the left descending coronary artery. Paraffin sections taken from infarcted areas of the left ventricle were stained with antibodies recognizing cardiomyocytes, neutrophils, macrophages and apoptotic cells. In both animal groups, a strong neutrophil response was noted in the infarcted myocardium, with a large proportion of these cells also displaying staining for anti-alpha-sarcomeric actin in the PAI-1KO animals. Abundant macrophages were also identified in the infarcted regions of both animal groups, forming demonstrable streams at the border region in the C57BL/6 control animals. Surprisingly, only sparse cells from both animal groups were labeled with the apoptotic markers anti-cleaved caspase 3 antibody and anti-single stranded DNA antibody (following formamide treatment). A dual immunostaining protocol was developed to localize both of these apoptotic markers in the same cell. Again, only scattered cells were found displaying both markers in the zones of infarction, suggesting that 3 days of persistent ischemia results in a robust necrotic response, but only a very minor apoptotic response in this mouse model.

Deleterious effects of lack of cardiac PAI-1 after coronary occlusion in mice and their pathophysiologic determinants.

We sought to delineate mechanisms through which the lack of plasminogen activator inhibitor (PAI)-1 in the heart affects remodeling of the heart early after myocardial infarction (MI). MI was induced by coronary occlusion in 10-weeks old PAI-1 knockout (KO) and control mice. Three days after MI, systolic and diastolic function was assessed with high-resolution echocardiography, infarct size was determined biochemically and histologically and accumulation of acute inflammatory cells in zones of infarction was characterized by immunocytochemistry. PAI-1 KO mice exhibited markedly thickened diastolic left ventricular anterior walls (1.38 +/- 0.38 mm vs. 0.77 +/- 0.13 SD), more profound depression of global and regional cardiac function (19 vs. 22% fractional shortening), and greater evidence of diastolic dysfunction (average E wave amplitude = 568 vs. 675 mm/s) all of which were significant. Markedly greater extent of infarction was demonstrated biochemically and histologically in knockout mice compared with controls (76 vs. 29% of the left ventricle, P < 0.05) associated with striking hemorrhage and intense inflammation. Fibrosis normalized for infarct size was markedly reduced (0.006 vs. 0.022 microg hydroxyproline/mg dry weight). Thus, lack of PAI-1 in the heart exerted deleterious effects mediated, at least in part by increased inflammation and hemorrhage and attenuating of fibrosis.

Salutary effects of attenuation of angiotensin II on coronary perivascular fibrosis associated with insulin resistance and obesity.

Obesity and insulin resistance confer increased risk for accelerated coronary disease and cardiomyopathic phenomena. We have previously shown that inhibition of angiotensin-converting enzyme (ACE) prevents coronary perimicrovascular fibrosis in genetically obese mice that develop insulin resistance. This study was performed to elucidate mechanism(s) implicated and to determine the effects of attenuation of angiotensin II (Ang) II. Genetically obese ob/ob mice were given ACE inhibitor (temocapril) or Ang II type 1 (AT(1)) receptor blocker (olmesartan) from 10 to 20 weeks. Cardiac expressions of plasminogen activator inhibitor (PAI)-1, the major physiologic inhibitor of fibrinolysis, and transforming growth factor (TGF)-beta(1), a prototypic profibrotic molecule, were determined and extent of perivascular coronary fibrosis was measured. Twenty-week-old obese mice exhibited increased plasma levels of PAI-1 and TGF-beta(1) compared with the values in lean counterpart. Perivascular coronary fibrosis in arterioles and small arteries was evident in obese mice that also showed increased left ventricular collagen as measured by hydroxyproline assay. Immunohistochemistry confirmed the deposition of perivascular type 1 collagen. Markedly increased PAI-1 and TGF-beta were seen immunohistochemically in coronary vascular wall and confirmed by western blotting. When obese mice were treated with temocapril or olmesartan from 10 to 20 weeks, both were equally effective and prevented increases in perivascular fibrosis, plasma PAI-1 and TGF-beta(1), left ventricular collagen and mural immunoreactivity for PAI-1, TGF-beta and collagen type 1. The c-Jun NH(2)-terminal kinase (JNK) activity was elevated in the left ventricle of obese mice (western) and blocked by temocapril and olmesartan. Ang II-mediated upregulation of PAI-1 and TGF-beta(1) with collagen deposition may explain the mechanism of perivascular fibrosis in obese mice. ACE inhibition and blockade of AT(1) receptor may prevent coronary perivascular fibrosis and collagen deposition even before development of overt diabetes. JNK activation may be a mediator of obesity-related cardiac dysfunction and a potential therapeutic target.

Coronary capillary network remodeling and hypofibrinolysis in aged obese diabetic rats: implications for increased myocardial vulnerability to ischemia.

Despite the known abnormalities of cardiac function in patients with overt non-insulin dependent diabetes mellitus (NIDDM) the temporal changes of coronary capillary network remodeling leading to potential microcirculatory dysfunction have not been elucidated. To this end, left ventricular subendocardial capillary network of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, characterized by hypertension, obesity, hyperglycemia, hyperinsulinemia and mild NIDDM, and control Long-Evans Tokushima (LETO) rats were investigated. Total capillary density in OLETF was significantly higher than that in LETO at 20 weeks, suggesting compensatory improvement of O2 transport at early stages of NIDDM. The increase in capillary density in OLETF was lost at 40 and 60 weeks due to the decreases of intermediate capillary portions and venular capillary portions. Although capillary domain area (area innervated by single capillary) in OLETF was lower than that in LETO at 20 weeks, the values were similar between OLETF and LETO at 40 and 60 weeks, suggesting that adaptive improvement in the capacity for 02 transport with a high perfusion was lost in late stages of NIDDM. Activity of plasma plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of proteo(fibrino)lysis, in OLETF was higher than that in LETO at 40 and 60 weeks, suggesting that increase of PAI-1 may downregulate compensatory adaptive capillary network remodeling by inhibiting proteolysis and angiogenesis in the cardiac interstitium. Loss of adaptive myocardial microcirculation may therefore contribute to increased vulnerability in ischemic injury and to cardiac dysfunction in NIDDM.