Lerman, Amir

Background and Purpose— Adipose tissue is an abundant source of endothelial cells as well as stem and progenitor cells which can develop an endothelial phenotype. It has been demonstrated that these cells have distinct angiogenic properties in vitro and in vivo. However, whether these cells have the capacity to directly improve large vessel form and function after vascular injury remains unknown. To define whether delivery of adipose-derived endothelial cells (ADECs) would improve healing of injured carotid arteries, a rabbit model of acute arterial injury was used. Methods— Autologous rabbit ADECs were generated using defined culture conditions. To test the ability of ADECs to enhance carotid artery repair, cells were delivered intraarterially after acute balloon injury. Additional delivery studies were performed after functional selection of cells before delivery. Results— After rabbit omental fat harvest and digestion, a proliferative, homogenous, and distinctly endothelial population of ADECs was identified. Direct delivery of autologous ADECs resulted in marked reendothelialization 48 hours after acute vascular injury as compared to saline controls (82.2±26.9% versus 4.2±3.0% P <0.001). Delivery of ADECs that were selected for their ability to take up acetylated LDL significantly improved vasoreactivity and decreased intimal formation after vascular injury. Conclusions— Taken together, these data suggest that ADECs represent an autologous source of proliferative endothelial cells, which demonstrate the capacity to rapidly improve reendothelialization, improve vascular reactivity, and decrease intimal formation in a carotid artery injury model.

Background— Endothelial progenitor cells (EPCs) promote neovascularization and endothelial repair. Renal artery stenosis (RAS) may impair renal function by inducing intrarenal microvascular injury and remodeling. We investigated whether replenishment with EPCs would protect the renal microcirculation in chronic experimental renovascular disease. Methods and Results— Single-kidney hemodynamics and function were assessed with the use of multidetector computed tomography in vivo in pigs with RAS, pigs with RAS 4 weeks after intrarenal infusion of autologous EPCs, and controls. Renal microvascular remodeling and angiogenic pathways were investigated ex vivo with the use of micro–computed tomography, histology, and Western blotting. EPCs increased renal expression of angiogenic factors, stimulated proliferation and maturation of new vessels, and attenuated renal microvascular remodeling and fibrosis in RAS. Furthermore, EPCs normalized the blunted renal microvascular and filtration function. Conclusions— The present study shows that a single intrarenal infusion of autologous EPCs preserved microvascular architecture and function and decreased microvascular remodeling in experimental chronic RAS. It is likely that restoration of the angiogenic cascade by autologous EPCs involved not only generation of new vessels but also acceleration of their maturation and stabilization. This contributed to preserving the blood supply, hemodynamics, and function of the RAS kidney, supporting EPCs as a promising therapeutic intervention for preserving the kidney in renovascular disease.

Endothelin plays an important role in the pathogenesis of atherosclerosis. The aim of the study was to evaluate the safety and hemodynamic and metabolic responses to 6 months treatment with atrasentan, the selective endothelin-A receptor antagonist. Seventy-two patients with multiple cardiovascular risk factors and nonobstructive coronary artery disease on coronary angiogram were randomly assigned in a double-blind manner to atrasentan or placebo. Mean aortic blood pressure decreased from 92±10 to 80±10 mm Hg ( P <0.001) in the atrasentan group and did not change in the placebo group (93±10 and 92±11 mm Hg; P =0.84). The difference between the groups was significant ( P <0.001). No effect on heart rate was observed. In a subgroup of patients not treated with angiotensin-converting enzyme inhibitor, creatinine level decreased in the atrasentan versus the placebo group ( P =0.011). Fasting glucose ( P =0.026), glycosylated hemoglobin level ( P =0.041), triglyceride l ( P =0.013), lipoprotein-A ( P =0.046), and uric acid levels ( P =0.048) decreased significantly in the atrasentan group compared with the placebo group. No progression of angiographic coronary disease was observed. The most common adverse effects with atrasentan were nasal stuffiness, headache, and edema. In conclusion, 6 months of treatment with atrasentan results in a reduction of blood pressure and improvement in glucose and lipid metabolism. These findings suggest the beneficial role of atrasentan in the treatment of hypertension and metabolic syndrome.

Background and Purpose— Circulating lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) has emerged as a novel biomarker for cardiovascular diseases. However, the correlation between the plaque expression of Lp-PLA 2 and plaque oxidative stress, inflammation, and stability as well as the clinical presentation remains poorly defined, especially for cerebrovascular disease. Therefore, this study was performed to test the hypothesis that Lp-PLA 2 expression is higher in symptomatic than in asymptomatic carotid plaques of patients undergoing carotid endarterectomy. Methods— The expression of Lp-PLA 2 in 167 carotid artery plaques was determined by immunoblotting and immunostaining. Plaque oxidative stress, inflammation, and stability were quantified by NAD(P)H oxidase p67phox and MMP-2 immunoblotting, oxidized LDL (oxLDL) immunoreactivity, macrophage and Sirius red collagen staining. Lysophosphatidylcholine 16:0 (lysoPC) concentration was measured in 55 plaques using liquid chromatography tandem mass spectrometry. Results— Lp-PLA 2 expression was significantly higher in plaques of symptomatic patients than asymptomatic patients (1.66±0.19 versus 1.14±0.10, P <0.05) and localized mainly to shoulder and necrotic lipid core areas in colocalization with oxLDL and macrophage content. Similarly, Lp-PLA 2 expression was related to collagen content, which was lower in plaques from symptomatic patients than in plaques from asymptomatic patients (9.1±2.2 versus 18.5±1.7% of staining/field, P <0.001). LysoPC plaque concentration was significantly higher in plaques of symptomatic than asymptomatic patients (437.0±57.91 versus 228.84±37.00 mmol/L, P <0.05). Conclusions— Symptomatic carotid artery plaques are characterized by increased levels of Lp-PLA 2 and its product lysoPC in correlation with markers of tissue oxidative stress, inflammation, and instability. These findings strongly support a role for Lp-PLA2 in the pathophysiology and clinical presentation of cerebrovascular disease.

In vitro and animal studies suggest that oxidative stress is associated with endothelial dysfunction. We tested whether local oxidative stress and nitric oxide (NO) bioavailability in the coronary circulation is associated with coronary endothelial dysfunction in humans. Blood samples were obtained simultaneously from the left main coronary artery and the coronary sinus for measurement of F2-isoprostanes, myeloperoxidase, nitrotyrosine, and superoxide dismutase in 20 patients without significant coronary disease. Afterward, coronary blood flow and the vascular response to intracoronary acetylcholine and N G -monomethyl- L -arginine (L-NMMA) were assessed. The gradient of isoprostanes between the arterial levels and coronary sinus correlated with the change in coronary artery diameter in response to acetylcholine ( r =−0.79, P <0.0001). Isoprostanes net production across the left anterior descending artery territory correlated with a decrease in superoxide dismutase activity ( r =0.66, P =0.002) and decrease in coronary artery diameter in response to L-NMMA (r s =0.48, P <0.05). Myeloperoxidase and nitrotyrosine gradients were similar in patients with endothelial dysfunction and controls. The effect of L-NMMA was similar in both groups. We conclude that coronary endothelial dysfunction in humans is characterized by local enhancement of oxidative stress without a decrease in basal NO release. This study supports the hypothesis that local oxidative stress has a role in reduction of NO bioavailability in humans with coronary endothelial dysfunction.

Background— We investigated the potential of conversion to sirolimus (SRL) as a primary immunosuppressant in attenuating cardiac allograft vasculopathy progression. Methods and Results— Twenty-nine cardiac transplant recipients were converted to SRL 3.8±3.4 years after transplantation with complete calcineurin inhibitor (CNI) withdrawal. Secondary immunosuppressants (azathioprine or mycophenolate) and steroids remained unchanged. Forty patients (controls) 4.8±4.0 years from transplantation were maintained on CNIs. Three-dimensional intravascular ultrasound studies were performed at baseline and 12.1±2.6 months later. Mean plaque (media and intima) volume (PV) and plaque index (PI) (PV/vessel volume percent) increased significantly in the CNI group (1.28±2.86 mm 3 /mm, P =0.004; and 6±8%, P =0.0001) but not in the SRL group (0.1±1.13 mm 3 /mm, P =0.63; and 0.1±8%, P =0.94). In patients enrolled within 2 years after transplantation, the increases in PV (0.06±1.06 versus 1.77±1.65 mm 3 /mm; P =0.0081) and PI (0±9% versus 10±8%; P =0.0145) were smaller in the SRL group (n=11) than in the CNI (n=12) group. In patients enrolled ≥2 years after transplantation, the increase in PI was less in the SRL group compared with the CNI group (0.1±6.5% versus 5±8%; P =0.033), but changes in PV did not differ significantly. Treatment with azathioprine or mycophenolate did not affect PV or PI in either the SRL group (PV: 0.22±0.66 versus 0.05±1.45 mm 3 /mm, P =0.46; PI: 1.5±6% versus −1.6±8.5%, P =0.29) or the CNI group (PV: 1.42±1.39 versus 1.06±2.28 mm 3 /mm, P =0.49; PI: 7.8±8.7% versus 4.8±7.3%, P =0.23). Conclusions— Substituting CNI with SRL as primary immunosuppression attenuates cardiac allograft vasculopathy progression.

Endothelial cells play important structural and functional roles in vascular homoeostasis. Perturbations in endothelial cell number and function are directly involved with the initiation and progression of multiple cardiovascular diseases, including atherosclerosis, hypertension and congestive heart failure. Attempts to modify these disorders have included pharmacological strategies to improve vascular and thus endothelial function. A goal of biological approaches to these disorders is the delivery of endothelial cells that might act to provide beneficial endothelial-derived factors. However, this approach has generally been limited by the lack of readily available autologous endothelial cells for delivery. The isolation of circulation-derived endothelial progenitor cells allows for direct access to autologous endothelial cells for preclinical and clinical studies. Preclinical studies using autologous endothelial cells have demonstrated beneficial effects when delivered in animal models of vascular injury and grafting. These effects are related to the endothelial nature of the cells and may be paracrine in nature. Ongoing studies are aimed at defining the nature of these effects and optimizing delivery strategies cognizant of these mechanisms.

The purpose of this work was to compare the effects of hypertension and hypercholesterolemia on carotid endothelial function, structure, and vasa vasorum density. Seventeen pigs were randomized to a 12-week normal diet without (n=5), or with renovascular hypertension (HT; n=6), or to a high cholesterol diet (HC; n=6). Carotid arteries were studied by organ chambers (endothelial function) and microcomputed tomography (vasa vasorum), and tissue was processed for Sirius red staining and immunoblotting (vascular endothelium growth factor, endostatin, matrix metalloproteinase-9, and matrix metalloproteinase-2). HC and HT showed reduced vasodilation to acetylcholine as compared with controls, but HT also had a lower response to sodium nitroprusside. In addition, HT showed a higher content of organized collagen fibers and increased intima-media thickness. Vasa vasorum density was increased in HC but not in HT. Both HT and HC showed a proangiogenetic biochemical milieu (higher vascular endothelium growth factor, matrix metalloproteinases, and lower endostatin), but this was more pronounced in HC. Both hypertension and hypercholesterolemia induce endothelial dysfunction in the carotid artery. However, hypertension is also associated with greater fibrosis and vascular wall thickening, which might impair endothelium-independent vasorelaxation and vasa vasorum growth. Hypercholesterolemia is, in turn, associated with vasa vasorum neovascularization. These data suggest that carotid atherosclerosis can evolve through different mechanisms in relation to different risk factors.

The proteasome is responsible for the degradation of oxidized proteins, and proteasome inhibition has been shown to generate oxidative stress in vitro. Atherosclerosis is thought to be initiated as a consequence of increased endogenous oxidative stress. The current study was designed to assess whether chronic proteasome inhibition is associated with early coronary atherosclerosis. Female pigs, 3 months of age, were randomized to a normal (N) or high-cholesterol (HC) diet (2% cholesterol, 15% lard) without or with twice weekly subcutaneous injections of the proteasome inhibitor (PSI) MLN-273 (0.08 mg/kg, N+PSI and HC+PSI) for a period of 12 weeks (n=5 per group). Coronary vasorelaxation to bradykinin (10 −10.5 to 10− 6.5 mol/L) and sodium nitroprusside (10 −9 to 10 −5 mol/L) was assessed by in vitro organ chamber experiments, intima–media ratio by morphometric analysis of Elastica–van Gieson–stained slides, and intima superoxide production by dihydroethidium fluorescence. Vasorelaxation to 10 −6.5 mol/L bradykinin was reduced in HC compared with N (69±7 versus 90±2%, P <0.05) and further reduced in N+PSI and HC+PSI (57±6 and 48±13%, P <0.05 versus N and HC for each). Compared with N (0.03±0.01), intima–media ratio was higher in N+PSI (0.09±0.04, P <0.01) and HC+PSI (0.15±0.06, P <0.05). Compared with N (0.6±0.9% of intima area), dihydroethidium fluorescence was higher in HC, N+PSI, and HC+PSI (8.9±1.6, 6.0±3.5, and 7.2±3.9% of intima area, P <0.05 for all). Thus, chronic proteasome inhibition is associated with increased coronary artery oxidative stress and early atherosclerosis. These findings support the significance of the proteasome and related protein quality control for vascular biology and pathology.