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Objectives This study hypothesized that compensatory mitral leaflet area (MLA) adaptation occurs in patients with persistent atrial fibrillation (AF) without left ventricular (LV) dysfunction but has limitations that augment mitral regurgitation (MR). The study also explored whether asymmetrical annular dilation is matched by relative leaflet enlargement. Background Functional MR occurs in patients with AF and isolated annular dilation, but the relationship of MLA adaptation with annular area (AA) is unknown. Methods Three-dimensional echocardiographic images were acquired from 86 patients with quantified MR: 53 with nonvalvular persistent AF (23 MR+ with moderate or greater MR, 30 MR−) without LV dysfunction or dilation and 33 normal controls. Comprehensive 3-dimensional analysis included total diastolic MLA, adaptation ratios of MLA to annular area and MLA to leaflet closure area, and annular and tenting geometry. Results Total MLA was 22% larger in patients with AF than in controls, thus paralleling the increased AA. However, as AA increased, adaptive indices (MLA/AA ratio and ratio of MLA to closure area) plateaued, becoming lowest in MR+ patients (ratio of MLA to closure area = 1.63 ± 0.17 controls, 1.60 ± 0.11 MR−, 1.32 ± 0.10 MR+; p Conclusions MLA adaptively increases in AF with isolated annular dilation and normal LV function. This compensatory enlargement becomes insufficient with greater annular dilation, and the leaflets fail to match asymmetrical annular remodeling, thereby increasing MR. These findings can potentially help optimize therapeutic options and motivate basic studies of adaptive growth processes.

Sturge-Weber syndrome (SWS) is a vascular neurocutaneous disorder that results from a somatic mosaic mutation in GNAQ, which is also responsible for isolated port-wine birthmarks. Infants with SWS are born with a cutaneous capillary malformation (port-wine birthmark) of the forehead or upper eyelid which can signal an increased risk of brain and/or eye involvement prior to the onset of specific symptoms. This symptom-free interval represents a time when a targeted intervention could help to minimize the neurological and ophthalmologic manifestations of the disorder. This paper summarizes a 2015 SWS workshop in Bethesda, Maryland that was sponsored by the National Institutes of Health. Meeting attendees included a diverse group of clinical and translational researchers with a goal of establishing research priorities for the next few years. The initial portion of the meeting included a thorough review of the recent genetic discovery and what is known of the pathogenesis of SWS. Breakout sessions related to neurology, dermatology, and ophthalmology aimed to establish SWS research priorities in each field. Key priorities for future development include the need for clinical consensus guidelines, further work to develop a clinical trial network, improvement of tissue banking for research purposes, and the need for multiple animal and cell culture models of SWS.

Thickening of mitral leaflets, endothelial-to-mesenchymal transition (EndMT), and activated myofibroblast-like interstitial cells have been observed in ischemic mitral valve regurgitation. We set out to determine if interactions between mitral valve endothelial cells (VECs) and interstitial cells (VICs) might affect these alterations. We used in vitro co-culture in Transwell™ inserts to test the hypothesis that VICs secrete factors that inhibit EndMT and conversely, that VECs secrete factors that mitigate the activation of VICs to a myofibroblast-like, activated phenotype. Primary cultures and clonal populations of ovine mitral VICs and VECs were used. Western blot, quantitative reverse transcriptase PCR (qPCR) and functional assays were used to assess changes in cell phenotype and behavior. VICs or conditioned media from VICs inhibited transforming growth factor β (TGFβ)-induced EndMT in VECs, as indicated by reduced expression of EndMT markers α-smooth muscle actin (α-SMA), Slug, Snai1 and MMP-2 and maintained the ability of VECs to mediate leukocyte adhesion, an important endothelial function. VECs or conditioned media from VECs reversed the spontaneous cell culture-induced change in VICs to an activated phenotype, as indicated by reduced expression of α-SMA and type I collagen, increased expression chondromodulin-1 (Chm1), and reduced contractile activity. These results demonstrate that mitral VECs and VICs secrete soluble factors that can reduce VIC activation and inhibit TGFβ-driven EndMT, respectively. These findings suggest that the endothelium of the mitral valve is critical for the maintenance of a quiescent VIC phenotype and that, in turn, VICs prevent EndMT. We speculate that the disturbance of the ongoing reciprocal interactions between VECs and VICs in vivo may contribute to the thickened and fibrotic leaflets observed in ischemic mitral regurgitation, and in other types of valve disease.

SummaryEndothelial dysfunction is a central hallmark of diabetes. The transcriptional coactivator PGC-1α is a powerful regulator of metabolism, but its role in endothelial cells remains poorly understood. We show here that endothelial PGC-1α expression is high in diabetic rodents and humans and that PGC-1α powerfully blocks endothelial migration in cell culture and vasculogenesis in vivo. Mechanistically, PGC-1α induces Notch signaling, blunts activation of Rac/Akt/eNOS signaling, and renders endothelial cells unresponsive to established angiogenic factors. Transgenic overexpression of PGC-1α in the endothelium mimics multiple diabetic phenotypes, including aberrant re-endothelialization after carotid injury, blunted wound healing, and reduced blood flow recovery after hindlimb ischemia. Conversely, deletion of endothelial PGC-1α rescues the blunted wound healing and recovery from hindlimb ischemia seen in type 1 and type 2 diabetes. Endothelial PGC-1α thus potently inhibits endothelial function and angiogenesis, and induction of endothelial PGC-1α contributes to multiple aspects of vascular dysfunction in diabetes.

Background Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo. Methods Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence. Results Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein–positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein–positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein–expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein–positive endothelial progenitor cells. Conclusions The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.

The interaction and interplay between endothelial cells (ECs) and mural cells (such as vascular smooth muscle cells -VSMCs- and pericytes) play a pivotal role in vascular biology. Endoglin is an RGD-containing ligand of β1 integrins highly expressed by ECs during angiogenesis. Our working hypothesis is that endothelial endoglin acts as an adhesion molecule via integrin recognition motifs, allowing the interaction between ECs and mural cells. We find that suppression of endoglin expression or addition of soluble endoglin inhibits the adhesion between ECs and VSMCs as shown by tubulogenesis assays on matrigel. The EC-VSMC adhesion was also abolished by an antiintegrin α5β1 inhibitory antibody, whereas it was markabsedly enhanced by the integrin activators MnCl2 or CXCL12. The CXCL12-dependent cell adhesion was abolished in the presence of soluble endoglin or a derived pentapeptide containing the RGD motif. Adhesion of cells overexpressing different endoglin mutant constructs, allowed the specific mapping of the endoglin RGD motif as involved in adhesion to VSMCs. Binding of soluble endoglin to VSMCs was markedly enhanced by MnCl2 and CXCL12 and this increase was inhibited by the RGD peptide. Moreover, transgenic mice overexpressing soluble endoglin show podocyturia and lower number of glomerular podocytes, suggesting that soluble endoglin induced the detachment of podocytes from glomerular capillaries. These results suggest a critical role for endoglin in integrin- mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel development and maturation in normal physiology as well as in pathologies such as preeclampsia, cancer or hereditary hemorrhagic telangiectasia.

Background This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach. Methods Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing. Results Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including α-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets. Conclusions Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form "tissue" that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.

Infantile hemangioma is a benign tumor of endothelial cells (ECs) that is well known to pediatricians because of its frequency and sometimes disturbingly rapid growth. The primary defect(s) that cause hemangioma have not yet been elucidated, but recent studies have revealed that hemangiomas are composed of clonal ECs that exhibit abnormal properties in vitro. These studies support the hypothesis that hemangiomas arise when a somatic mutation occurs in a single endothelial progenitor, leading to dysregulated activity of one or more genes that control EC growth. Aberrant endothelial growth may in turn alter patterns of gene expression in nearby cells--in the overlying epidermis, for example--and account for observed extrinsic alterations that may influence hemangiogenesis. Unraveling the cellular and molecular basis of hemangioma is likely to yield critical insights into the mechanisms of EC proliferation and regression that may be applicable to other cardiovascular disorders.

Human AC133 antigen, also called CD133, was recently identified as a hematopoietic stem cell marker. However, the molecular structure and function of this protein has remained unclear. Here we cloned and identified a novel isoform of AC133, which we named AC133-2. In comparison to the reported AC133 cDNA, which is referred to herein as AC133-1, a small exon of 27 nucleotides is deleted in AC133-2 by alternative mRNA splicing. Similar to the previously characterized AC133 antigen, recombinant AC133-2 expressed in 293 cells was glycosylated and transported to plasma membrane. AC133-2 mRNA was found predominant in a variety of human fetal tissue, adult tissues, and several carcinomas. In contrast, AC133-1 mRNA was more prominent in fetal brain and adult skeletal muscle but was not detected in fetal liver and kidney, adult pancreas, kidney, and placenta, suggesting different roles for the two isoforms in fetal development and mature organ homeostasis. Here, we demonstrate that AC133-2 is the isoform expressed on hematopoietic stem cells derived from fetal liver, bone marrow, and peripheral blood. The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells. To further investigate its expression in other stem cell populations, we found that AC133-2 co-expressed with beta(1) integrin in the basal layer of human neonatal epidermis. AC133-2(+)/beta(1) integrin(+) cells proliferated and differentiated in culture, which coincided with a loss of AC133-2 and gain in a terminal differentiation marker involucrin. Taken together, these results suggest that AC133-2 is expressed in multiple stem cell niches and may provide a means to isolate specific stem cell subpopulations from human tissues.

Cardiac valves arise from endocardial cushions, specialized regions of the developing heart that are formed by an endothelial-to-mesenchymal cell transdifferentiation. Whether and to what extent this transdifferentiation is retained in mature heart valves is unknown. Herein we show that endothelial cells from mature valves can transdifferentiate to a mesenchymal phenotype. Using induction of alpha-smooth muscle actin (alpha-SMA), an established marker for this process, two distinct pathways of transdifferentiation were identified in clonally derived endothelial cell populations isolated from ovine aortic valve leaflets. alpha-SMA expression was induced by culturing clonal endothelial cells in medium containing either transforming growth factor-beta or low levels of serum and no basic fibroblast growth factor. Cells induced to express alpha-SMA exhibited markedly increased migration in response to platelet-derived growth factor-BB, consistent with a mesenchymal phenotype. A population of the differentiated cells co-expressed CD31, an endothelial marker, along with alpha-SMA, as seen by double-label immunofluorescence. Similarly, this co-expression of endothelial markers and alpha-SMA was detected in a subpopulation of cells in frozen sections of aortic valves, suggesting the transdifferentiation may occur in vivo. Hence, the clonal populations of valvular endothelial cells described here provide a powerful in vitro model for dissecting molecular events that regulate valvular endothelium.

Capillaries provide a vast interface between the blood and the tissues that is crucial for regulating nutrient delivery, blood coagulation and transmigration of leukocytes to sites of infection. The growth of new capillaries from pre-existing vessels (angiogenesis) is essential for normal embryogenesis and growth, but also occurs in the development of many diseases. Although relatively little is known about endothelial cell biology, progress is nevertheless being made towards understanding angiogenesis, and several laboratories have begun to identify cell adhesion molecules that may be required for the growth of microvessels.

A cDNA encoding a homologue of human P-selectin has been isolated from a bovine capillary endothelial cDNA library. The 2.7 kb cDNA encodes a 646 amino acid polypeptide with 77% identity to the human P-selectin except that it lacks three of the consensus repeat domains found in human P-selectin. Human P-selectin, expressed in platelets and endothelium, is a Ca 2+ -dependent receptor for myeloid cells that binds to carbohydrates on neutrophils and monocytes. To determine if bovine P-selectin exhibits a similar binding activity, its cDNA was expressed in COS cells and the ability of the transfectants to bind HL-60 human myelogenous leukemia cells was examined. The bovine P-selectin bound the myeloid cells in a manner similar to human P-selectin, indicating that the altered domain structure of bovine P-selectin does not affect P-selectin function in this in vitro cell adhesion assay.

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.

Prostacyclin therapy has markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, so the aim of our study was to determine how treprostinil contributes to the angiogenic functions of endothelial progenitors (ECFC). Treprostinil did not modify clonogenic properties nor endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. Silencing or blocking VEGF-A in MSC blocked the pro-angiogenic effect of treprostinil in vitro and in vivo. Clinical relevance was confirmed by the high level of VEGF-A detected in plasma from patients with pediatric pulmonary hypertension who had been treated with treprostinil. Our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy

The mannose analogue, 1-deoxymannojirimycin, has been tested for its effect on five alpha-mannosidase activities present in rat liver and shown to be a specific inhibitor of Golgi alpha-mannosidase I at low mumolar concentrations. Golgi alpha-mannosidases I and II were assayed in a highly purified Golgi membrane preparation. Endoplasmic reticulum alpha-mannosidase activity was measured in a rough endoplasmic reticulum detergent extract. A purified soluble alpha-mannosidase activity which we believe is derived from the endoplasmic reticulum during tissue homogenization was also tested. And finally, the lysosomal or acidic alpha-mannosidase was measured in a postnuclear supernatant fraction obtained from rat liver. The results presented here show that 1-deoxymannojirimycin inhibits only Golgi alpha-mannosidase I, which is consistent with its effect on oligosaccharide processing in vivo (Fuhrmann et al. Nature 1984 307:755-758).