Background— Cell therapy with bone marrow–derived stem/progenitor cells is a novel option for improving neovascularization and cardiac function in ischemic heart disease. Circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. However, whether this impairment also extends to bone marrow–derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) is unclear. Methods and Results— BM-MNCs were isolated from bone marrow aspirates in 18 patients with ICMP (ejection fraction, 38±11%) and 8 healthy control subjects (controls). The number of hematopoietic stem/progenitor cells (CD34 + /CD133 + ), CD49d + (VLA-4) cells, and CXCR4 + cells did not differ between the 2 groups. However, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls (37.3±25.0 versus 113.8±70.4 granulocyte-macrophage colony-forming units; P =0.009). Likewise, the migratory response to stromal cell–derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls (SDF-1, 46.3±26.2 versus 108.6±40.4 cells/microscopic field, P <0.001; VEGF, 34±24.2 versus 54.8±29.3 cells/microscopic field, P =0.027). To assess the in vivo relevance of these findings, we tested the functional activity of BM-MNCs to improve neovascularization in a hindlimb animal model using nude mice. Two weeks after ligation of the femoral artery and intravenous injection of 5×10 5 BM-MNCs, laser Doppler–derived relative limb blood flow in mice treated with BM-MNCs from patients with ICMP was significantly lower compared with mice treated with BM-MNCs from healthy controls (0.45±0.14 versus 0.68±0.15; P <0.001). The in vivo neovascularization capacity of BM-MNCs closely correlated with the in vitro assessment of SDF-1–induced migration ( r =0.78; P <0.001) and colony-forming capacity ( r =0.74; P <0.001). Conclusions— BM-MNCs isolated from patients with ICMP have a significantly reduced migratory and colony-forming activity in vitro and a reduced neovascularization capacity in vivo despite similar content of hematopoietic stem cells. This functional impairment of BM-MNCs from patients with ICMP may limit their therapeutic potential for clinical cell therapy.

Thus, elevated CRP serum levels indicative of a systemic inflammatory response are associated with a blunted systemic endothelial vasodilator function. The identification of elevated CRP levels as a transient independent risk factor for endothelial dysfunction might provide an important clue to link a systemic marker of inflammation to atherosclerotic disease progression.

Background-Primary and secondary prevention trials suggest that statins possess favorable effects independent of cholesterol reduction. We investigated whether statin therapy may also accelerate reendothelialization after carotid balloon injury. Methods and Results-Simvastatin treatment in 34 male Sprague-Dawley rats accelerated reendothelialization of the balloon-injured arterial segments (reendothelialized area at 2 weeks, 12.3Ϯ1.8 versus 5.4Ϯ1.1 mm 2 , PϽ0.01) and resulted in a dose-dependent (0.2 or 1 mg/kg IP) significant reduction in neointimal thickening at 2, 3, and 4 weeks compared with saline-injected controls (nϭ18). To elucidate the mechanism, we investigated the contribution of bone marrow-derived endothelial progenitor cells (EPCs) by bone marrow transplantation from Tie2/lacZ mice to background mice or nude rats. X-gal staining of mouse carotid artery specimens revealed a 2.9-fold increase in the number of ␤-gal-positive cells per square millimeter appearing on the carotid artery luminal surface at 2 weeks, and double-fluorescence immunohistochemistry disclosed a significant 5-fold increase in the number of double-positive cells (␤-gal, isolectin B4) on the luminal surface in carotid arteries of statin-treated nude rats (20Ϯ3 versus 4Ϯ1 cells/mm surface length, PϽ0.005). Statins increased circulating rat EPCs (2.4-fold at 2 weeks and 2.5-fold at 4 weeks, PϽ0.001) and induced adhesiveness of cultured human EPCs by upregulation of the integrin subunits ␣ 5 , ␤ 1 , ␣ v , and ␤ 5 of human EPCs as shown by reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting. Conclusions-These findings establish additional mechanisms by which statins may specifically preempt disordered vascular wall pathology and constitute physiological evidence that EPC mobilization represents a functionally relevant consequence of statin therapy.

Abstract-Transplantation of bone marrow cells as well as circulating endothelial progenitor cells (EPC) enhancesneovascularization after ischemia. The chemokine receptor CXCR4 is essential for migration and homing of hematopoietic stem cells. Therefore, we investigated the role of CXCR4 and its downstream signaling cascade for the angiogenic capacity of cultured human EPC. Ex vivo, differentiated EPC derived from peripheral blood abundantly expressed CXCR4. Incubation of EPC from healthy volunteers with neutralizing antibodies against CXCR4 profoundly inhibited vascular endothelial growth factor-and stromal-derived factor-1-induced migration as well as EPC-induced angiogenesis in an ex vivo assay. Preincubation of transplanted EPC with CXCR4 antibody reduced EPC incorporation and impaired blood-flow recovery in ischemic hindlimbs of nude mice (57Ϯ4% of normal perfusion versus untreated EPC: 80Ϯ11%, PϽ0.001). Bone marrow mononuclear cells (BM-MNC) or EPC of heterozygous CXCR4 ϩ/Ϫ mice displayed reduced CXCR4 expression and disclosed impaired in vivo capacity to enhance recovery of ischemic blood flow in nude mice (blood flow 27Ϯ11% versus 66Ϯ25% using wild-type cells, PϽ0.01). Importantly, impaired blood flow in ischemic CXCR4 ϩ/Ϫ mice was rescued by injection of wild-type BM-MNC. Next, we investigated the role of CXCR4 for functional capacities of EPC from patients with coronary artery disease (CAD). Surface expression of CXCR4 was similar in EPC from patients with CAD compared with healthy controls. However, basal Janus kinase (JAK)-2 phosphorylation was significantly reduced and less responsive to stromal-derived factor-1 in EPC from patients with CAD compared with healthy volunteers, indicating that CXCR4-mediated JAK-2 signaling is dysregulated in EPC from patients with CAD. The CXCR4 receptor signaling profoundly modulates the angiogenic activity and homing capacity of cultured human EPC. Disturbance of CXCR4 signaling, as demonstrated by reduced JAK-2 phosphorylation, may contribute to functional impairment of EPC from patients with CAD. Stimulating CXCR4 signaling might improve functional properties of EPC and may rescue impaired neovascularization capacity of EPC derived from patients with CAD. (Circ Res. 2005;97:1142-1151.) Key Words: coronary artery disease Ⅲ endothelium Ⅲ angiogenesis Ⅲ EPC C irculating endothelial progenitor cells (EPC) play a crucial role in postnatal neovascularization. 1-3 Increasing evidence suggests that transplantation of cultureexpanded progenitor cells or bone marrow-derived progenitor cells successfully promotes therapeutic neovascularization in both ischemic hindlimbs as well as acute myocardial infarction models. 4 -7 Moreover, recent clinical pilot studies suggest that not only restoration of blood flow in peripheral artery disease but also functional regeneration and left ventricular remodeling can be enhanced after autologous transplantation of bone marrow-derived cells or cultured EPC in patients with coronary atherosclerosis. 8 -11 Further evidence indicates that no...

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Background— Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking. Methods and Results— Forty patients with critical limb ischemia were included in a multicenter, phase II, double-blind, randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and, thus, the trial missed its primary end point. However, cell therapy was associated with significantly improved ulcer healing (ulcer area, 3.2±4.7 cm 2 to 1.89±3.5 cm 2 [ P =0.014] versus placebo, 2.92±3.5 cm 2 to 2.89±4.1 cm 2 [ P =0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [ P =0.009] versus placebo, 4.5±2.4 to 3.9±2.6 [ P =0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups. Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage ( r =0.8; P <0.001). Conclusions— Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00282646.

Background-Transplantation

Both the processes of atherosclerosis and plaque rupture are indicated to be influenced by matrix metalloproteinase (MMP) activity. We therefore searched for common functional variation in the matrix metalloelastase (MMP-12) gene locus that may be implicated in coronary artery disease. Single-strand conformation polymorphism analysis of DNA from healthy individuals detected a common polymorphism within the MMP-12 gene promoter (an A-to-G substitution at position -82). The frequency of the G allele was 0. 19. The polymorphism influences the binding of the transcription factor activator protein-1 (AP-1) in electromobility shift assay. A higher binding affinity of AP-1 to the A allele was associated with higher MMP-12 promoter activity in vitro in transient transfection studies in U937 and murine lung macrophage (MALU) cells. Phorbol 12-myristate 13-acetate (PMA) and insulin, 2 known activators of AP-1, increased the binding of AP-1 to the MMP-12 promoter, with higher affinity for the A allele. In transfection experiments, both the A and the G alleles responded to insulin and PMA, the A allele showing higher promoter activity than the G allele. Furthermore, Western blot analysis demonstrated that insulin increased MMP-12 protein production. To analyze whether the -82 A/G polymorphism is associated with coronary artery disease, 367 consecutive patients who underwent percutaneous transluminal coronary angiography with stent implantation were genotyped. In patients (n=71) with diabetes, the A allele was associated with a smaller luminal diameter. In conclusion, a common functional polymorphism within the MMP-12 promoter influences coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease.