Christian Ihling

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3−/−) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3−/− mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3−/− mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3−/− mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.

Tissue Endothelin-1 Immunoreactivity in the Active Coronary Atherosclerotic Plaque A Clue to the Mechanism of Increased Vasoreactivity of the Culprit Lesion in Unstable Angina

BACKGROUND The pathomorphological substrate of complicated coronary atherosclerotic lesions underlying unstable angina is characterized by a localized chronic inflammatory process. Functionally, coronary lesions associated with unstable angina demonstrate an enhanced vasoreactivity. Endothelin-1 is a potent vasoconstrictor peptide produced not only by endothelial cells but also by macrophages and polymorphonuclear leukocytes, the cell types characteristic of inflammation. METHODS AND RESULTS By use of immunohistochemical techniques, we examined the presence of endothelin-1 in coronary atherosclerotic plaque tissue obtained by directional coronary atherectomy of primary lesions from 50 consecutive patients. The tissue specimens of 43 of 50 patients (86%) demonstrated endothelin-1-like immunoreactivity. Endothelin-1-like immunoreactivity preferentially localized to macrophage-rich areas, to hypercellular regions rich in microvessels, and to plaque areas with evidence of prior hemorrhage. Double-immunolabeling revealed that both macrophages (HAM56 positive) and intimal smooth muscle cells (alpha-actin positive) demonstrated cytoplasmic immunostaining for endothelin-1. Semiquantitative analysis of endothelin-1-like immunostaining revealed significantly (P < .005) higher staining grades in active (1.86 +/- 0.15, n = 40) compared with nonactive lesions (0.78 +/- 0.35, n = 10): endothelin-1 staining grades were significantly (P < .001) lower in patients with stable angina (0.69 +/- 0.19, n = 13) than in patients with crescendo angina (1.82 +/- 0.30, n = 11), with angina at rest (2.08 +/- 0.21, n = 12), or with angina after myocardial infarction (2.0 +/- 0.26, n = 14). CONCLUSIONS Endothelin-1 immunostaining of atherosclerotic tissue localizes predominantly with plaque components indicative of chronic inflammatory processes. The increased tissue endothelin-1-like immunoreactivity in active coronary atherosclerotic lesions may provide a clue to the mechanisms of increased vasoreactivity of the culprit lesion in acute ischemic syndromes, which is the clinical substrate of the active coronary atherosclerotic plaque.

Expression of Macrophage Migration Inhibitory Factor in Different Stages of Human Atherosclerosis

Background—Atherosclerosis is a chronic inflammatory response of the arterial wall to injury. Macrophage migration inhibitory factor (MIF), a cytokine with potent inflammatory functions, was thus considered to be important in atherosclerotic lesion evolution. Methods and Results—We studied the presence and distribution of MIF immunoreactivity (MIF-IR) and MIF mRNA in internal mammary arteries with a normal histology and arteries with plaques in different stages of human atherosclerosis. To address a potential role for the coactivator Jab1 as a cellular mediator of MIF effects in vascular tissue, we correlated the expression of MIF to that of Jab1 by using immunohistochemistry and coimmunoprecipitation. We further sought to determine a potential functional role for endothelium-derived MIF in early atherogenesis by studying the effects of oxidized LDL on MIF expression in cultured human umbilical vascular endothelial cells. The results showed that MIF-IR and Jab1-IR are found in all cell types present in atherosclerotic lesions, that MIF-IR is upregulated during progression of atherosclerosis, that MIF is produced locally in the arterial wall, and that all MIF+ cells are simultaneously Jab1+. Coimmunoprecipitation experiments demonstrated in vivo complex formation between MIF and Jab1 in plaques. MIF expression in human umbilical vascular endothelial cells and a macrophage line was upregulated after stimulation with oxidized LDL. Conclusions—MIF is produced abundantly by various cells in all types of human atherosclerotic lesions and thus may play an important role in early plaque development and advanced complicated lesions. MIF-Jab1 complexes could serve critical regulatory functions in atherosclerotic lesion evolution.

Fas receptor signaling inhibits glycogen synthase kinase 3β and induces cardiac hypertrophy following pressure overload

Congestive heart failure is a leading cause of mortality in developed countries. Myocardial hypertrophy resulting from hypertension often precedes heart failure. Understanding the signaling underlying cardiac hypertrophy and failure is of major interest. Here, we identified Fas receptor activation, a classical death signal causing apoptosis via activation of the caspase cascade in many cell types, as a novel pathway mediating cardiomyocyte hypertrophy in vitro and in vivo. Fas activation by Fas ligand induced a hypertrophic response in cultured cardiomyocytes, which was dependent on the inactivation of glycogen synthase kinase 3 beta (GSK3 beta) by phosphorylation. In vivo, lpr (lymphoproliferative disease) mice lacking a functional Fas receptor demonstrated rapid-onset left ventricular dilatation and failure, absence of compensatory hypertrophy, and significantly increased mortality in response to pressure overload induction that was accompanied by a failure to inhibit GSK3 beta activity. In contrast, Fas ligand was dispensable for the development of pressure overload hypertrophy in vivo. In vitro, neonatal cardiomyocytes from lpr mice showed a completely abrogated or significantly blunted hypertrophic response after stimulation with Fas ligand or angiotensin II, respectively. These findings indicate that Fas receptor signaling inhibits GSK3 beta activity in cardiomyocytes and is required for compensation of pressure overload in vivo.

Sustained Persistence of Transplanted Proangiogenic Cells Contributes to Neovascularization and Cardiac Function After Ischemia

Circulating blood–derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Induction of cell death in the previously transplanted cells reduced perfusion and led to vascular leakage as evidenced by Evans blue extravasation. These results indicate that physical incorporation and persistence of cells contribute to cell-mediated improvement of neovascularization and cardiac function. Long-term paracrine activities and/or cell intrinsic mechanisms may have contributed to the maintenance of functional improvement.

Co-expression of p53 and MDM2 in human atherosclerosis : Implications for the regulation of cellularity of atherosclerotic lesions

Atherosclerosis is a fibroproliferative disease of the arterial intima. It was recently found that wild‐type p53 (wt p53) accumulates in human atherosclerotic tissue. Wt p53 is a cell cycle regulator involved in DNA repair, DNA synthesis, cell differentiation, and apoptosis and might therefore make an important contribution to the cellularity of atherosclerotic plaques. The product of the MDM2 gene is a nuclear protein which forms a complex with p53, thereby inhibiting the negative regulatory effects of wt p53 on cell cycle progression. In order to address a potential role of the interaction of p53 with MDM2 for the regulation of cellularity in atherosclerotic tissue, 22 carotid atheromatous plaques from patients undergoing endarterectomy were studied to determine the presence of p53 immunoreactivity (IR), MDM2 IR, cell proliferation as evidenced by MIB1/Ki‐67 IR and DNA fragmentation by in situterminal transferase‐mediated dUTP 3′ end labelling (TUNEL), as a marker for apoptosis. p53 IR localized to areas with evidence of chronic inflammation (22/22) and was observed in virtually all cell types in 68·79±7·51 per cent of the nuclei. p53 staining in the control tissue from human internal mammary arteries was present in 0·2±0·29 per cent of the cells (P≤0·002). MDM2 IR was present in all cases (22/22) in macrophages and smooth muscle cells (SMCs) in 60·53±8·32 per cent of the nuclei (controls: 0·8±0·65 per cent, P≤0·002) and co‐localized with p53 IR as shown by examination of adjacent sections and by double immunofluorescence labelling. Importantly, co‐immunoprecipitation and western blot analysis revealed that p53 and MDM2 were physically associated, indicating that MDM2–p53 complex formation takes place in vivoin human atherosclerotic tissue. Positive TUNEL staining and MIB1/Ki‐67 IR present in 3·01±1·27 per cent of the nuclei (controls: 0 per cent, P≤0·002) localized to the same plaque compartments as p53 IR and MDM2 IR. Thus, the fate of cells with p53 accumulation may depend on the interaction and the stoichiometry of the p53 and MDM2 proteins. Cells were indeed found with strong p53 accumulation and nuclear morphology typical for apoptosis and there were a few MIB1/Ki‐67‐positive cells with co‐expression of MDM2, indicating a possible role for MDM2 in reversing the negative regulatory effects of p53 for cell cycle progression. The nuclear co‐localization of p53 IR with MDM2 IR and the co‐immunoprecipitation assay indicate the presence of p53–MDM2 complex formation in vivo in human atherosclerotic tissue. The destiny of individual p53 and MDM2‐co‐expressing cells either to undergo p53‐dependent apoptosis or to re‐enter the cycle of cell proliferation may depend on the relative ratios of the two proteins. p53 and MDM2 may therefore play an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques.

Topographical Association Between the Cyclin-Dependent Kinases Inhibitor P21, p53 Accumulation, and Cellular Proliferation in Human Atherosclerotic Tissue

The cell cycle is controlled by cyclin-dependent protein kinases (CDKs). The activity of these enzymes is directed by inhibitors of CDKs. The 21-kD protein product (P21) of the WAF1/CIP1 gene, which can be transactivated by the protein product of the tumor suppressor gene p53, acts as an inhibitor of cyclin-dependent kinases. To assess whether both P21 and p53 may play a role in the control of cellular proliferation in atherosclerotic lesions, the topographical association between p53, P21, and the proliferation marker MIB1/Ki-67, was analyzed by immunohistochemistry in human carotid atheromatous plaques of 26 patients. p53 immunoreactivity (IR) was present in 26 of 26 cases in the nuclei of virtually all cell types (macrophages [MPs], smooth muscle cells [SMCs], endothelial cells [ECs]) in areas with chronic inflammation in 71.08 +/- 8.28% of the nuclei. p53 staining in the control tissue from human coronary arteries was present in 0.3 +/- 0.45% of the cells (P < .002): P21-IR was present in 24 of 26 specimens in 64.38 +/- 10.13% of the cells (controls: 3.8 +/- 1.85%, P < .002) and localized to nuclei of MPs (CD68 positive) and SMCs (alpha-actin positive), as well as ECs of microvessels present in 21 specimens (21 of 21) and luminal ECs present in 18 specimens (16 of 18). As shown by double labeling, P21-IR colocalized with p53-IR in most MPs (24 of 24), intimal SMCs (22 of 24), ECs of microvessels (19 of 21), and luminal ECs (10 of 16). Interestingly, few p53-positive cells did not show simultaneous P21-IR, and, conversely, not all P21-positive cells demonstrated p53-IR. MIB1/Ki-67-positive cells were identified in 21 of 26 tissue specimens in 3.53 +/- 1.79% of the nuclei (controls: 0%, P < .002) and localized principally to MPs bordering the atheromatous lipid core (21 of 26) and to a few scattered SMCs (16 of 26), ECs of microvessels (13 of 21), and luminal ECs (2 of 18). Most importantly, none of the cells coexpressing P21 and p53 were positive for MIB1/Ki-67-IR, indicating the absence of proliferating activity. In summary, this study demonstrates that P21-IR is present in the atherosclerotic plaque and colocalizes with p53 in most MPs, SMCs, and ECs. The lack of proliferation markers in cells coexpressing p53 and P21 suggests that transcriptional activation of the WAF1/CIP1 gene by p53 may be involved in the control of cellular proliferation in advanced human atherosclerotic plaques.

Inhibition of endogenous nitric oxide synthase potentiates ischemia–reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway

OBJECTIVE Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

The Wnt Antagonist Dickkopf-1 Mobilizes Vasculogenic Progenitor Cells via Activation of the Bone Marrow Endosteal Stem Cell Niche

Therapeutic mobilization of vasculogenic progenitor cells is a novel strategy to enhance neovascularization for tissue repair. Prototypical mobilizing agents such as granulocyte colony-stimulating factor mobilize vasculogenic progenitor cells from the bone marrow concomitantly with inflammatory cells. In the bone marrow, mobilization is regulated in the stem cell niche, in which endosteal cells such as osteoblasts and osteoclasts play a key role. Because Wnt signaling regulates endosteal cells, we examined whether the Wnt signaling antagonist Dickkopf (Dkk)-1 is involved in the mobilization of vasculogenic progenitor cells. Using TOP-GAL transgenic mice to determine activation of β-catenin, we demonstrate that Dkk-1 regulates endosteal cells in the bone marrow stem cell niche and subsequently mobilizes vasculogenic and hematopoietic progenitors cells without concomitant mobilization of inflammatory neutrophils. The mobilization of vasculogenic progenitors required the presence of functionally active osteoclasts, as demonstrated in PTPϵ-deficient mice with defective osteoclast function. Mechanistically, Dkk-1 induced the osteoclast differentiation factor RANKL, which subsequently stimulated the release of the major bone-resorbing protease cathepsin K. Eventually, the Dkk-1–induced mobilization of bone marrow–derived vasculogenic progenitors enhanced neovascularization in Matrigel plugs. Thus, these data show that Dkk-1 is a mobilizer of vasculogenic progenitors but not of inflammatory cells, which could be of great clinical importance to enhance regenerative cell therapy.

Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects

OBJECTIVES The aim of this study was to investigate whether the caspase-3 inhibitor Ac-DEVD-CHO functionally improves stunned myocardium. BACKGROUND Degradation of troponin I contributes to the pathogenesis of myocardial stunning, whereas the role of apoptosis is unknown. Caspase-3 is an essential apoptotic protease that is specifically inhibited by Ac-DEVD-CHO. METHODS Isolated working hearts of rats were exposed to 30 min of low-flow ischemia, followed by 30 min of reperfusion. Ac-DEVD-CHO (0.1 to 1 micromol/l) was added 15 min before ischemia/reperfusion or 5 min before reperfusion. Cardiac output, external heart power, left ventricular (LV) developing pressure and contractility (dp/dt(max)) were measured. Apoptosis was assessed by TUNEL staining and internucleosomal deoxyribonucleic acid fragmentation. Caspase-3 processing and troponin I cleavage were determined by immunoblotting. Caspase-3 activity was measured using a fluorogenic substrate. RESULTS The addition of Ac-DEVD-CHO before ischemia/reperfusion or before reperfusion dose-dependently and significantly (p < 0.05) improved post-ischemic recovery of cardiac output, external heart power, LV developing pressure and dp/dt(max), compared with the vehicle (0.01% dimethyl sulfoxide). Ac-DEVD-CHO was similarly effective when given before reperfusion. Ac-DEVD-CHO blocked ischemia/reperfusion-induced caspase-3 activation, but cardiomyocyte apoptosis was unaffected. Troponin I cleavage was not inhibited by Ac-DEVD-CHO. CONCLUSIONS Caspase-3 is activated in stunned myocardium. Inhibition of caspase-3 by Ac-DEVD-CHO significantly improves post-ischemic contractile recovery of stunned myocardium, even when given after the onset of ischemia. The mechanism(s) of protection by Ac-DEVD-CHO appear to be independent of apoptosis. Inhibition of caspase-3 is a novel therapeutic strategy to improve functional recovery of stunned myocardium.