Imo E. Hoefer

Exenatide Reduces Infarct Size and Improves Cardiac Function in a Porcine Model of Ischemia and Reperfusion Injury

OBJECTIVES This study sought to examine whether exenatide is capable of reducing myocardial infarct size. BACKGROUND Exenatide is a glucagon-like peptide (GLP)-1 analogue with insulinotropic and insulinomimetic properties. Because insulin and GLP-1 have been described as reducing apoptosis, exenatide might confer cardioprotection after acute myocardial infarction (MI). METHODS Pigs were randomized to exenatide or phosphate-buffered saline (PBS) treatment after 75 min of coronary artery ligation and subsequent reperfusion. Infarct size was assessed with Evans Blue (Sigma-Aldrich, St. Louis, Missouri) and triphenyltetrazolium chloride. Cardiac function was measured with epicardial ultrasound and conductance catheter-based pressure-volume loops. Western blotting, histology, and activity assays were performed to determine markers of apoptosis/survival and oxidative stress. RESULTS Exenatide reduced myocardial infarct size (32.7 +/- 6.4% vs. 53.6 +/- 3.9%; p = 0.031) and prevented deterioration of systolic and diastolic cardiac function (systolic wall thickening: 47.3 +/- 6.3% vs. 8.1 +/- 1.9%, p < 0.001; myocardial stiffness: 0.12 +/- 0.06 mm Hg/ml vs. 0.22 +/- 0.07 mm Hg/ml; p = 0.004). After exenatide treatment, myocardial phosphorylated Akt and Bcl-2 expression levels were higher compared with those after PBS treatment, and active caspase 3 expression was lower. In addition, fewer cells were terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling-positive. In addition, nuclear oxidative stress as assessed with an 8-hydroxydeoxyguanosine staining was reduced in the exenatide treatment arm, and superoxide dismutase activity and catalase activity were increased. Serum insulin levels increased after exenatide treatment, without affecting glucose levels. CONCLUSIONS These data identify exenatide as a potentially effective compound to reduce infarct size in adjunction to reperfusion therapy in patients with acute MI.

Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease

After birth two forms of vessel growth can be observed; angiogenesis and arteriogenesis. Angiogenesis refers to the formation of capillary networks. Arteriogenesis refers to the growth of preexistent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. The process of arteriogenesis is initiated when shear stresses increase in the preexistent collateral pathways upon narrowing of a main artery. The increased shear stress leads to an upregulation of cell adhesion molecules for circulating monocytes, which accumulate subsequently around the proliferating arteries and provide the several required cytokines and growth factors. Several strategies are currently tested for their potential to stimulate the process of arteriogenesis. These strategies focus either at shear stress, at direct stimulation of endothelial and smooth muscle cell growth or at the monocytic pathway and promising results were obtained from experimental studies. However, some important questions remain to be answered before arteriogenesis can be brought from bench to bedside.

Toll-Like Receptor 4 Mediates Maladaptive Left Ventricular Remodeling and Impairs Cardiac Function After Myocardial Infarction

Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction. Therapeutic options to prevent LV remodeling are limited, which necessitates the exploration of alternative therapeutic targets. Toll-like receptors (TLRs) serve as pattern recognition receptors within the innate immune system. Activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling following myocardial infarction. To establish the role of TLR4 in postinfarct LV remodeling, myocardial infarction was induced in wild-type BALB/c mice and TLR4-defective C3H-Tlr4LPS−d mice. Without affecting infarct size, TLR4 defectiveness reduced the extent of LV remodeling (end-diastolic volume: 103.7±6.8 &mgr;L versus 128.5±5.7 &mgr;L; P<0.01) and preserved systolic function (ejection fraction: 28.2±3.1% versus 16.6±1.3%; P<0.01), as assessed by MRI. In the noninfarcted area, interstitial fibrosis, and myocardial hypertrophy were reduced in C3H-Tlr4LPS−d mice. In the infarcted area, however, collagen density was increased, which was accompanied by fewer macrophages, reduced inflammation regulating cytokine expression levels (interleukin [IL]-1&agr;, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, tumor necrosis factor-&agr;, interferon-&ggr;, granulocyte/macrophage colony-stimulating factor), and reduced matrix metalloproteinase-2 (4684±515 versus 7573±611; P=0.002) and matrix metalloproteinase-9 activity (76.0±14.3 versus 168.0±36.2; P=0.027). These data provide direct evidence for a causal role of TLR4 in postinfarct maladaptive LV remodeling, probably via inflammatory cytokine production and matrix degradation. TLR4 may therefore constitute a novel target in the treatment of ischemic heart failure.

Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.

Myocardial Ischemia/Reperfusion Injury Is Mediated by Leukocytic Toll-Like Receptor-2 and Reduced by Systemic Administration of a Novel Anti–Toll-Like Receptor-2 Antibody

Background— Reperfusion therapy for myocardial infarction is hampered by detrimental inflammatory responses partly via Toll-like receptor (TLR) activation. Targeting TLR signaling may optimize reperfusion therapy and enhance cell survival and heart function after myocardial infarction. Here, we evaluated the role of TLR2 as a therapeutic target using a novel monoclonal anti-TLR2 antibody. Method and Results— Mice underwent 30 minutes of ischemia followed by reperfusion. Compounds were administered 5 minutes before reperfusion. Cardiac function and dimensions were assessed at baseline and 28 days after infarction with 9.4-T mouse magnetic resonance imaging. Saline and IgG isotype treatment resulted in 34.5±3.3% and 31.4±2.7% infarction, respectively. Bone marrow transplantation experiments between wild-type and TLR2-null mice revealed that final infarct size is determined by circulating TLR2 expression. A single intravenous bolus injection of anti-TLR2 antibody reduced infarct size to 18.9±2.2% (P=0.001). Compared with saline-treated mice, anti-TLR2–treated mice exhibited less expansive remodeling (end-diastolic volume 68.2±2.5 versus 76.8±3.5 &mgr;L; P=0.046) and preserved systolic performance (ejection fraction 51.0±2.1% versus 39.9±2.2%, P=0.009; systolic wall thickening 3.3±6.0% versus 22.0±4.4%, P=0.038). Anti-TLR2 treatment significantly reduced neutrophil, macrophage, and T-lymphocyte infiltration. Furthermore, tumor necrosis factor-&agr;, interleukin-1&agr;, granulocyte macrophage colony-stimulating factor, and interleukin-10 were significantly reduced, as were phosphorylated c-jun N-terminal kinase, phosphorylated p38 mitogen-activated protein kinase, and caspase 3/7 activity levels. Conclusions— Circulating TLR2 expression mediates myocardial ischemia/reperfusion injury. Antagonizing TLR2 just 5 minutes before reperfusion reduces infarct size and preserves cardiac function and geometry. Anti-TLR2 therapy exerts its action by reducing leukocyte influx, cytokine production, and proapoptotic signaling. Hence, monoclonal anti-TLR2 antibody is a potential candidate as an adjunctive for reperfusion therapy in patients with myocardial infarction.

Time course of arteriogenesis following femoral artery occlusion in the rabbit.

OBJECTIVE We examined the time course of arteriogenesis (collateral artery growth) after femoral artery ligation and the effect of monocyte chemoattractant protein-1 (MCP-1). METHODS New Zealand White rabbits received MCP-1 or phosphate buffered saline (PBS) for a 1-week period, either directly or 3 weeks after femoral artery ligation (non-ischemic model). A control group was studied with intact femoral arteries and another 1 min after acute femoral artery ligation. RESULTS Collateral conductance index significantly increased when MCP-1 treatment started directly after femoral artery ligation (acute occlusion: 0.94+/-0.19; without occlusion: 168.56+/-15.99; PBS: 4.10+/-0.48; MCP-1: 33.96+/-1.76 ml/min/100 mmHg). However, delayed onset of treatment 3 weeks after ligation and final study of conductance at 4 weeks showed no significant difference against a 4-week control (PBS: 79.08+/-7.24; MCP-1: 90.03+/-8.73 ml/min/100 mmHg). In these groups increased conductance indices were accompanied by a decrease in the number of visible collateral vessels (from 18 to 36 identifiable vessels at day 7 to about four at 21 days). CONCLUSION We conclude that the chemokine MCP-1 markedly accelerated collateral artery growth but did not alter its final extent above that reached spontaneously as a function of time. We show thus for the first time that a narrow time window exists for the responsiveness to the arteriogenic actions of MCP-1, a feature that MCP-1 may share with other growth factors. We show furthermore that the spontaneous adaptation by arteriogenesis stops when only about 50% of the vasodilatory reserve of the arterial bed before occlusion are reached. The superiority of few large arterial collaterals in their ability to conduct large amounts of blood flow per unit of pressure as compared to the angiogenic response where large numbers of small vessels are produced with minimal ability to allow mass transport of bulk flow is stressed.

GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function

We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocytes life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.

Arteriogenesis Proceeds via ICAM-1/Mac-1- Mediated Mechanisms

Monocyte adhesion to shear stress–activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte adhesion via intercellular adhesion molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, monocyte chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or MCP-1 plus an IgG2a isotype control via osmotic minipumps. After 7 days, collateral conductance was evaluated: solvent 4.01 (mL/min per 100 mm Hg), MCP-1 plus ICAM-mab 8.04 (versus solvent P =NS), and MCP-1 alone 33.11 (versus solvent P <0.05). Furthermore, the right femoral arteries of ICAM-1−/−, Mac-1−/− and mice having defective selectin interactions (FT4/7−/−) as well as their corresponding controls were ligated. One week later, perfusion ratios were determined by the use of fluorescent microspheres. FT4/7−/− mice did not show any significant difference in perfusion restoration whereas ICAM-1−/− and Mac-1−/− mice had a significant reduction in arteriogenesis as compared with matching controls (FT4/7-WT 37±9%, FT4/7−/− 32±3%, P =0.31; C57BL/6J 59±9%, ICAM-1−/− 36±8%, P <0.05; Mac-1−/− 42±3%, P <0.05). ICAM-1/Mac-1–mediated monocyte adhesion to the endothelium of collateral arteries is an essential step for arteriogenesis, whereas this process can proceed via selectin interaction independent mechanisms. Furthermore, in vivo treatment with monoclonal antibodies against ICAM-1 totally abolishes the stimulatory effect of MCP-1 on collateral artery growth, suggesting that the mechanism of the MCP-1–induced arteriogenesis proceeds via the localization of monocytes rather than the action of the MCP-1 molecule itself.

G-CSF administration after myocardial infarction in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis

Granulocyte‐colony stimulating factor (G‐CSF) has been shown to improve cardiac function after myocardial infarction (MI) by bone marrow cell mobilization and by protecting cardiomyocytes from apoptotic cell death. However, its role in collateral artery growth (arteriogenesis) has not been elucidated. Here, we investigated the effect of G‐CSF on arteriolar growth and cardiac function in a murine MI model.

Exogenous application of transforming growth factor beta 1 stimulates arteriogenesis in the peripheral circulation

Increased expression of transforming growth factor β1 (TGF‐ß1) during collateral artery growth, as well as its numerous effects on monocytes/macrophages and the smooth muscle cell cycle and differentiation, suggest a modulating role for this growth factor during arteriogenesis. We studied the effects of exogenously applied TGF‐ß1 on arteriogenesis as well as its interactions with monocytes, endothelial cells, and smooth muscle cells. In a New Zealand White (NZW) rabbit model of femoral artery ligation, increased expression of active TGF‐ß1 was found around proliferating arteries in NZW rabbits. The exogenous application of TGF‐ß1 led to an increase in both the number of visible collateral arteries as well as the conductance of the collateral circulation (4.0 ± 0.5 ml/min/100 mmHg vs. 28.9 ± 3.7 ml/min/100 mmHg, P < 0.05). Fluorescence activated cell sorting analysis showed an increase in the expression of the MAC‐1 receptor in both rabbit and human monocytes after treatment with TGF‐ß1 (control: 91.2 ± 4.2/482 ± 21.7; TGF‐ß1 200 ng/ml 193.9 ± 6.7/ 675.5 ± 25.7, P < 0.05 for all differences). TGF‐ß1 treated monocytes showed an increased endothelial adhesion and transmigration in transendothelial migration assays (5.75 ± 0.63 × 105 vs. 10.11 ± 0.04 × 105, P < 0.05). TGF‐ß1 had no direct pro‐angiogenic effect on human umbilical vein endothelial cells in a spheroid model of angiogenesis and inhibited the angiogenic effects of vascular endothelial growth factor.