Noel M. Caplice

Smooth Muscle Progenitor Cells in Human Blood

Background—Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs). Evidence for circulating SPCs in human subjects does not exist, and the mechanism whereby such putative SPCs may home to sites of plaque formation is presently not understood but is likely to involve expression of specific surface adhesion molecules, such as integrins. In this study, we aimed to culture smooth muscle outgrowth cells (SOCs) from SPCs in human peripheral blood and characterize surface integrin expression on these cells. Methods and Results—Human mononuclear cells isolated from buffy coat were seeded on collagen type 1 matrix and outgrowth cells selected in endothelial growth medium (EGM-2) or EGM-2 and platelet-derived growth factor BB. Selection in platelet-derived growth factor BB–enriched medium caused rapid outgrowth and expansion of SOC to >40 population doublings in a 4-month period. These SOCs were positive for smooth muscle cell–specific &agr; actin (&agr;SMA), myosin heavy chain, and calponin on immunofluorescence and Western blotting and were also positive for CD34, Flt1, and Flk1 receptor but negative for Tie-2 receptor expression, suggesting a potential bone marrow angioblastic origin. In contrast, endothelial outgrowth cells (EOCs) grown in EGM-2 alone and the initial MNC population were negative for these smooth muscle–specific markers. Integrin &agr;5&bgr;1 expression by FACS and Western blotting was significantly increased in SOCs compared with EOCs, and this was confirmed by 8-fold greater adhesion of SOC to fibronectin (P <0.001), an effect that could be decreased using an &agr;5&bgr;1 antibody. Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P <0.001). Conclusions—This study demonstrates for the first time outgrowth of smooth muscle cells with a specific growth, adhesion, and integrin profile from putative SPC in human blood. These data have implications for our understanding of adult vascular smooth muscle cell differentiation, proliferation, and homing. (Circulation. 2002;106:1199–1204.)

Bone Marrow–Derived Cardiomyocytes Are Present in Adult Human Heart A Study of Gender-Mismatched Bone Marrow Transplantation Patients

Background—Recent studies have identified cardiomyocytes of extracardiac origin in transplanted human hearts, but the exact origin of these myocyte progenitors is currently unknown. Methods and Results—Hearts of female subjects (n=4) who had undergone sex-mismatched bone marrow transplantation (BMT) were recovered at autopsy and analyzed for the presence of Y chromosome–positive cardiomyocytes. Four female gender-matched BMT subjects served as controls. Fluorescence in situ hybridization (FISH) for the Y chromosome was performed on paraffin-embedded sections to identify cells of bone marrow origin with concomitant immunofluorescent labeling for &agr;-sarcomeric actin to identify cardiomyocytes. A total of 160 000 cardiomyocyte nuclei were analyzed approximating 20 000 nuclei per patient. The mean percentage of Y chromosome–positive cardiomyocytes in patients with sex-mismatched BMT was 0.23±0.06%. Not a single Y chromosome–positive cardiomyocyte was identified in any of the control patients. Immunofluorescent costaining for laminin and chromosomal ploidy analysis with FISH showed no evidence of either pseudonuclei or cell fusion in any of the chimeric cardiac myocytes identified. Conclusions—These data establish for the first time human bone marrow as a source of extracardiac progenitor cells capable of de novo cardiomyocyte formation.

Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation

Atherosclerosis is the major cause of adult mortality in the developed world, and a significant contributor to atherosclerotic plaque progression involves smooth muscle cell recruitment to the intima of the vessel wall. Controversy currently exists on the exact origin of these recruited cells. Here we use sex-mismatched bone marrow transplant subjects to show that smooth muscle cells throughout the atherosclerotic vessel wall can derive from donor bone marrow. We demonstrate extensive recruitment of these cells in diseased compared with undiseased segments and exclude cell–cell fusion events as a cause for this enrichment. These data have broad implications for our understanding of the cellular components of human atherosclerotic plaque and provide a potentially novel target for future diagnostic and therapeutic strategies.

Human Endothelial Progenitor Cells Tolerate Oxidative Stress Due to Intrinsically High Expression of Manganese Superoxide Dismutase

Objective—Endothelial progenitor cells (EPCs) display a unique aptitude to promote angiogenesis and restore endothelial function of injured vessels. How progenitor cells can execute a regenerative program in the unfavorable environment of injury/inflammation-induced oxidative stress is poorly understood. We hypothesized that EPCs are resistant to oxidative stress and that this resistance is due to high expression and activity of antioxidant enzymes. Methods and Results—EPCs outgrown from human blood of healthy subjects demonstrated a marked resistance to cytotoxic effect of LY83583 (an O2&OV0254; generator), tumor necrosis factor-&agr;, and serum depletion. LY83583 inhibited in vitro tube formation by human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (CAECs), but not by EPCs. Compared with HUVECs and CAECs, EPCs exhibited ≈3- to 4-fold higher expression and activity of manganese superoxide dismutase (MnSOD), but not copper zinc superoxide dismutase (CuZnSOD) or catalase. The antioxidant profile in EPCs was associated with preservation of the mitochondrial network when exposed to LY83583. Moreover, cytotoxic effects of LY83583 on CAECs and HUVECs were reversed by adenoviral overexpression of MnSOD. Conclusions—Human EPCs are resistant to oxidative stress. High intrinsic expression of MnSOD is a critical mechanism protecting EPCs against oxidative stress.

Transplantation of Circulating Endothelial Progenitor Cells Restores Endothelial Function of Denuded Rabbit Carotid Arteries

Background and Purpose— Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries. Methods— Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (105 cells in 200 μL saline) or 200 μL saline alone (control) were administered into the lumen of injured artery. Results— Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall. Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5, P<0.05). Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells. Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines. Conclusions— We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function. Paracrine effects of EPCs may contribute to regenerative properties of EPCs.

Endothelial Progenitor Cells Are Decreased in Blood of Cardiac Allograft Patients With Vasculopathy and Endothelial Cells of Noncardiac Origin Are Enriched in Transplant Atherosclerosis

Background—Recent studies in animals suggest that circulating recipient endothelial precursors may participate in the biology of transplant vasculopathy. It is currently unknown whether a similar interaction between recipient endothelial cells and the vessel wall occurs in human subjects undergoing allogeneic cardiac transplantation. Methods and Results—Circulating endothelial cells and endothelial progenitor cells (EPCs) were quantified in 15 cardiac transplantation subjects with and without angiographic evidence of vasculopathy. In a separate series of experiments, the origin (donor or recipient) of transplant plaque endothelial cells was assessed in subjects who had undergone a gender-mismatched cardiac transplantation and had histological evidence of severe vasculopathy at the time of heart explantation. Circulating EPC outgrowth colonies in peripheral blood were significantly reduced in subjects with transplant vasculopathy compared with those without angiographic evidence of disease (EPC colony-forming units [CFUEPC]: 4.5±1.9 versus 15.1±3.7, P <0.05). There was no significant difference in circulating endothelial cell numbers as defined by day 4 culture acetylated LDL/lectin assay in either of these patient groups. In a separate group of 5 subjects who underwent gender-mismatched cardiac transplantation, there was a significant seeding of recipient endothelial cells (range: 1% to 24% of all luminal endothelial cells) in large-vessel lumen and adventitial microvessel lumen of arteriopathic vessels. No opposite-sex chimeric cells were observed in control gender-matched transplantation scenarios. Conclusions—These data suggest that the human cardiac transplant arteriopathy is associated with reduction in circulating endothelial precursors and with seeding of recipient-derived endothelial cells at the site of plaque development.

Dynamic Tracking During Intracoronary Injection of 18F-FDG-Labeled Progenitor Cell Therapy for Acute Myocardial Infarction

We assessed the feasibility of dynamic 3-dimensional (3D) PET/CT tracking of 18F-FDG-labeled circulating progenitor cell (CPC) therapy during intracoronary injection, using a porcine model of acute myocardial infarction (MI). Methods: Human and porcine CPC were radiolabeled with 18F-FDG, with variation in temperature and incubation time to determine optimal conditions. For in vivo experiments, CPC were harvested before induction of infarction (using 90-min coronary balloon occlusion). At 48 h, animals underwent cardiac MRI to assess infarct size. A balloon catheter was placed in the infarct artery at the same location as that used for induction of MI, and during dynamic 3D PET/CT 3 × 107 autologous 18F-FDG progenitor cells were injected through the central lumen using either (a) 3 cycles of balloon occlusion and reperfusion or (b) high-concentration, single-bolus injection without balloon occlusion (n = 3 for both protocols). Peripheral blood was drawn at 1-min intervals during cell injection. Results: Labeling efficiency was optimized by 30-min incubation at 37°C (human CPC, 89.9% ± 4.8%; porcine CPC, 91.6% ± 6.4%). Cell-bound activity showed a nonsignificant decrease at 1 h (human, 74.3% ± 10.7%; porcine, 77.7% ± 12.8%; P > 0.05) and a significant decrease at 2 h (human, 62.1% ± 8.9%; porcine, 68.6% ± 5.4%; P = 0.009). Mean infarct size was similar for both injection protocols (16.3% ± 3.4% and 20.6% ± 2.7%; P > 0.05). Dynamic scanning demonstrated a sharp rise in myocardial activity during each cycle of balloon-occlusion cell delivery, with a significant fall in activity (around 80%) immediately after balloon deflation. The latter was associated with a transient spike in peripheral blood 18F-FDG activity, consistent with the first pass of labeled cells in the systemic circulation. A single spike and gradual fall in myocardial activity was observed with high-concentration, single-bolus therapy. At 1 h, myocardial activity was 8.7% ± 1.5% of total injected dose for balloon-occlusion delivery and 17.8% ± 7.9% for high-concentration, single-bolus delivery (P = 0.08). Conclusion: Dynamic tracking during intracoronary injection of 18F-FDG-labeled CPC is feasible and demonstrates significant cell washout from the myocardium immediately after balloon deflation. High-concentration, single-bolus therapy may be as effective as balloon-occlusion delivery. This tracking technique should facilitate development of improved delivery strategies for cardiac cell therapy.

Activated nuclear factor-κB is present in the coronary vasculature in experimental hypercholesterolemia

BACKGROUND Experimental hypercholesterolemia (HC) is characterized by a decrease in nitric oxide (NO) bioavailability and cellular proliferation. Nuclear factor-kappaB (NF-kappaB) is a transcriptional factor which plays a coordinating role in inflammation and cellular proliferation and may be involved in early atherosclerosis. We examined whether activated NF-kappaB was present in experimental hypercholesterolemia in the coronary vasculature in association with a decrease in NO bioavailability. METHODS A total of 14 juvenile domestic crossbred pigs were placed on a HC diet and six pigs on a normal diet for 10-12 weeks. A monoclonal antibody to the activated form of the p65 subunit of NF-kappaB was used to detect immunoreactivity in coronary artery sections. Coronary tissue homogenates were analyzed for activated NF-kappaB and endothelial nitric oxide synthase (eNOS) using Western blotting. In vitro coronary endothelium-dependent relaxation was performed in response to bradykinin, as a measure of NO bioavailability. RESULTS Intimal staining for activated NF-kappaB was present in 12/14 HC pigs as compared with 0/6 controls (P<0.001). Confocal microscopy confirmed the presence of NF-kappaB in the nucleus of intimal cells although the majority of the staining was cytoplasmic. In the HC group, Western blotting revealed an increase in activated NF-kappaB in the vessel wall compared to the normal group, in association with a decrease in the presence of eNOS protein and an attenuated vasorelaxation response to bradykinin. CONCLUSION This study suggests a potential role for activation of NF-kappaB, in association with a decrease in NO bioavailability, in the initial stages of atherosclerosis in the coronary vasculature.

Antioxidant Intervention Attenuates Myocardial Neovascularization in Hypercholesterolemia

Background—Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1&agr; and vascular endothelial growth factor (VEGF). Methods and Results—Three groups of pigs (n= 6 each) were studied after 12 weeks of normal or 2% HC diet or HC+ antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1&agr;, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+ antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5± 11.8 versus 95.3± 8.2 vessels/cm2, P < 0.05), which was normalized in HC+ antioxidant (92.5± 20.5 vessels/cm2, P < 0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1&agr;, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention. Conclusions—Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1&agr; and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.

Clinical Potential of Adult Vascular Progenitor Cells

Cell therapy to treat vascular and cardiovascular diseases has evolved over the past decade with improved understanding of progenitor cell mobilization, recruitment, and differentiation. The beneficial effects seen in several preclinical studies have prompted translation of adult vascular progenitor therapy to clinical trials. To date, progenitor cells isolated from bone marrow and peripheral blood have been tested in the context of acute myocardial infarction and chronic ischemic cardiomyopathy, with moderate benefit. This therapeutic effect occurs despite a relatively small number of injected progenitor cells and short-term residence in the target zone. Thus, indirect benefits, such as paracrine factors released from these cells, have been suggested as significant contributors to therapeutic efficacy. Several additional vascular progenitors of endothelial, smooth muscle, mesenchymal, and cardiac origin have been identified that may contribute to vasculogenesis. Indeed, a unifying paradigm for the most effective cell therapy strategies to date appears to be robust support of angiogenesis. Here we discuss a number of progenitor cells that currently show potential as cardiovascular therapeutics, either singly or in combination. We look at emerging cell types and disease targets that may be exploited for therapeutic benefit and future strategies that may maximize clinical efficacy.