Abstract-Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascularresistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow-derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5Ϯ0.95 versus 48Ϯ3 mm Hg, respectively; PϽ0.001), whereas injection of skin fibroblasts had no protective effect (50.9Ϯ5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31Ϯ2 mm Hg, PϽ0.005) compared with day 21 (50Ϯ3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow-derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH. ( Key Words: progenitor cells Ⅲ pulmonary hypertension Ⅲ endothelium Ⅲ endothelial nitric oxide synthase P ulmonary arterial hypertension (PAH) is a devastating disease that in its most severe form, idiopathic PAH, leads to progressive debilitation and death, often within 2 to 3 years after its initial diagnosis. 1 Despite significant advances in the therapy of PAH during the last decade, the prognosis remains poor. Although the genetic basis for some patients with familial PAH has been elucidated, 2-4 how these mutations are causally linked to the development of PAH remains unclear. 5,6 Evidence from experimental models as well as lung specimens from patients with PAH underlines the importance of microvascular occlusion in the pathogenesis of this disease, 7 especially in its advanced stages; however, the precise mechanisms...

Background-Endothelium-derived nitric oxide (NO) is produced by an oxidative reaction catalyzed by endothelial NO synthase (eNOS). NO plays a crucial role in controlling cell growth and apoptosis, as well as having well-characterized vasodilator and antithrombotic actions. More recently, endothelium-derived NO was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. Therefore, we investigated the role of endothelium-derived NO during cardiovascular development using mice deficient in eNOS. Methods and Results-We examined the hearts of 12 mature eNOS-deficient and 26 mature wild-type mice. Five of the mature eNOS-deficient mice had a bicuspid aortic valve; none of the 26 wild-type animals exhibited identifiable valvular or cardiac abnormalities. Immunohistochemical analysis revealed prominent eNOS expression localized to the endothelium lining the valve cusps of the aorta in mature wild-type mice; expression was localized to the myocardium and endothelial cell monolayer lining the valve leaflets in the developing embryo. Conclusions-These results show a strong association between eNOS deficiency and the presence of a bicuspid aortic valve; they provide the first molecular insight into one of the most common types of congenital cardiac abnormality. Key Words: nitric oxide Ⅲ endothelium Ⅲ mice, knockout Ⅲ heart defects, congenital Ⅲ valves N itric oxide (NO) is a free radical gas that is synthesized from L-arginine in a complex oxidative reaction catalyzed by 3 distinct isoforms of NO synthase: endothelial (eNOS), inducible (iNOS), and neuronal (nNOS). 1 The neuronal isoform is highly expressed in neuronal cells and skeletal muscle, whereas the endothelial isoform is predominantly expressed in endothelial cells and produces small amounts of NO in response to intimal shear stress or agonists such as bradykinin. 2,3 The inducible isoform is expressed in many cell types, including macrophages and smooth muscle cells, primarily in response to inflammatory cytokines. Once expressed, this isoform can produce large amounts of NO in a continuous manner. 1,2 Endothelium-derived NO is crucial in the regulation of cell growth and apoptosis; it also has a well-characterized role as a vasodilator and antithrombotic agent. 1 More recently, NO derived from the endothelium was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. 4 -6 The valve leaflets of the heart originate from mesenchymal outgrowths known as cardiac cushions. Cushion formation is localized to the atrioventricular canal and ventricular outflow tract regions of the primary heart tube. These formations arise from regional thickenings of the cardiac jelly, the extracellular matrix that resides between the myocardium and endocardium of the primitive heart tube. This event involves the transformation of a subset of endothelial cells of the endocardium into mesenchyme. The mo...

There is evidence to suggest that the cellular components of homografts and bioprosthetic xenografts may contribute to calcification or immunogenic reactions. A four-step detergent and enzymatic extraction process has been developed to remove cellular components from bovine pericardial tissue. The process results in an acellular matrix material consisting primarily of elastin, insoluble collagen, and tightly bound glycosaminoglycans. Light and electron microscopy confirmed that nearly all cellular constituents are removed without ultrastructural evidence of damage to fibrous components. Collagen denaturation temperatures remained unaltered. Biochemical analysis confirmed the retention of collagen and elastin and some differential extraction of glycosaminoglycans. Low strain rate fracture testing and high strain rate viscoelastic characterization showed that, with the exception of slightly increased stress relaxation, the mechanical properties of the fresh tissue were preserved in the pericardial acellular matrix. Crosslinking of the material in glutaraldehyde or poly(glycidyl ether) produced mechanical changes consistent with the same treatments of fresh tissue. The pericardial acellular matrix is a promising approach to the production of biomaterials for heart valve or cardiovascular patching applications.

Abstract-Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor ␣, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 g of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (Pϭ0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Mice are widely used to study arterial disease in humans, and the pathogenesis of arterial diseases is known to be strongly influenced by hemodynamic factors. It is, therefore, of interest to characterize the hemodynamic environment in the mouse arterial tree. Previous measurements have suggested that many relevant hemodynamic variables are similar between the mouse and the human. Here we use a combination of Doppler ultrasound and MRI measurements, coupled with numerical modeling techniques, to characterize the hemodynamic environment in the mouse aortic arch at high spatial resolution. We find that the hemodynamically induced stresses on arterial endothelial cells are much larger in magnitude and more spatially uniform in the mouse than in the human, an effect that can be explained by fluid mechanical scaling principles. This surprising finding seems to be at variance with currently accepted models of the role of hemodynamics in atherogenesis and the known distribution of atheromatous lesions in mice.

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The infusion of freshly cultured allogenic bone marrow-derived MSCs, up to a dose of 3 million cells/kg (250 million cells), into participants with septic shock seems safe. Clinical trial registered with www.clinicaltrials.gov (NCT02421484).

Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.