Multiple combinations of melanocytic and vascular endothelial markers enhance the detection rate of lymphovascular invasion in cutaneous melanoma.

BACKGROUND: Lymphovascular invasion (LVI) is believed to be the mechanism by which melanoma cells can disseminate to regional lymph nodes and distant sites and may be predictive of adverse outcome. Lymphovascular invasion often difficult to detect on hematoxylin-eosin (HE) stained sections, are readily identified with dual immunohistochemistry (IHC) for melanocytic and vascular markers. METHODS: A total of 100 primary cutaneous malignant melanoma cases that had a Breslow thickness of 1-4 mm and lacked LVI by conventional HE assessment were included. We compared the LVI detection rates of double staining for CD31/S100 and CD34/S100, and D2-40/S100, and examined the association of LVI with clinical outcomes. RESULTS: The dual immunohistochemical positivity for CD31/S100, CD34/S100, and D2-40/S100 were 40(40%), 17(17%) and 35(35%), respectively. On multivariate analysis, LVI was an independent predictor of SLN status. Multivariate analysis revealed that LVI and male gender were independent risk factors for overall survival. CONCLUSIONS: The recognition of LVI is improved by dual IHC and predicts SLN metastasis. The detection of LVI using dual IHC, especially by a combination of CD31/S100 and D2-40/S100 is a useful step that inclusion should be recommended in basic evaluation parameters for cutaneous melanoma.

Dark-field microscopy enhance visibility of CD31 endothelial staining.

A simple dark field microscopy technique was used for visualization of blood vessels in normal human renal tissues and carcinoma. Phase contrast condenser ring apt for high power objectives was combined with a 10x objective in order to create a dark field illumination of the specimens examined. The endothelial lining of the vessels had been stained by using CD31 monoclonal antibodies combined with conventional peroxidase immunohistochemistry. The final DAB addition used for this technique induced an intense light scatter in the dark field microscope. This scattered light originating from the endothelial lining made the walls of the bright vessels easily detectable from the dark background.

Human CD31 on Swine Endothelial Cells Induces SHP-1 Phosphorylation in Macrophages.

BACKGROUND: Innate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. METHODS: SECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. RESULTS: No suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. CONCLUSIONS: Human CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

The vasa vasorum reach deep into the human thoracic aorta.

Only limited data are available on the extent of the vasa vasorum of the human thoracic aorta, although this could be important with regard to certain pathophysiological states, i.e. aortic aneurysm or atherosclerosis. A preliminary investigation shows that the vascularization of the human thoracic aorta reaches deeper layers than generally believed.

Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency.

Karaca N, Azarsiz E, Akarcan SE, Aksu G, Kütükçüler N. Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency. Turk J Pediatr 2019; 61: 885-894. Thymic maturation evaluation is inevitable for patients with clinical and laboratory findings for a primary immunodeficiency, as the T cellimmunodeficiencies are the most severe type. In this study, we aimed to show the usage of T cell surface molecule `CD31` for the evaluation of thymic output in patients (n: 66) with a large spectrum of findings signing a probable primary immunodeficiency. Besides the classical clinical and laboratory approach for these patients, T cell subpopulations as naive, memory, recent thymic emigrant cells were also investigated. The humoral immunodeficiency (34.8%), combined immunodeficiency (34.8%) and cardiopathy (7.6%) were the most frequent diagnosis groups. CD4+CD45RA+ naive T-cells percentages (p: 0.011) and absolute counts (p: 0.004) and absolute CD4+CD45RA+CD31+ RTE (recent thymic emigrant) cell counts (p: 0.007) were significantly lower in combined immunodeficiency group. Naive T-cells (p: 0.037) and RTE cells (p: 0.032) were also lower in patients who had cardiac surgery in the past. In conclusion, flow cytometric CD31+thymic naive RTE cell evaluation may provide rapid clinical information especially on T-cell immune dysfunction and CD4+CD45RA+CD31+ RTE cells may be used as an alternative to TRECs in the diagnosis of combined immunodeficiencies.

Isolation of tumor endothelial cells from murine cancer.

Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting. The high purity of the obtained cells was verified by flow cytometry. Upon cell culture, the isolated cells showed a polygonal shape and a cobblestone appearance, which are features of the endothelial cells. Furthermore, a functional assay revealed that the TECs suppressed the proliferation of CD8+ T cells in vitro. We believe that the isolation method described in this study will enable the further elucidation of the characteristics of TECs.

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

The Majority of CD45- Ter119- CD31- Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors.

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45- Ter119- CD31- (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51- TNCs. Ly6D+ CD44+ CD51- TNCs phenotypically and functionally resembled CD45+ pro-B lymphoid cells, whereas Ly6D- CD44+ CD51- TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44+ CD51- TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44+ CD51- TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45- hematopoietic progenitors.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44+Lin- and CD44-Lin- subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44+Lin- compared to CD44-Lin- cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

Human CD31 on porcine cells suppress xenogeneic neutrophil-mediated cytotoxicity via the inhibition of NETosis.

BACKGROUND: Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil-targeted therapies may have the potential for increased graft survival in xenotransplantation. METHODS: A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. RESULTS: A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1. CONCLUSION: These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.

Cellular atypia is negatively correlated with immunohistochemical reactivity of CD31 and vWF expression levels in canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) is one of the most common mesenchymal tumors in dogs. Its high metastatic and growth rates are usually associated with poor prognosis. Neoplastic cells of HSA can show various levels of cellular atypia in the same mass and may consist of various populations at different differentiated stages. Up to present, however, there is no report analyzing their differentiation states by comparing cellular atypia with differentiation-related protein expressions. To evaluate whether cellular atypia can be used as a differentiation marker in HSA, we analyzed correlation between cellular atypia and intensities of CD31 and von Willebrand Factor (vWF) staining in HSA cases. We also compared cellular atypia and expression levels of CD31 and vWF in each growth patterns. Our results show that cellular atypia was negatively correlated to CD31 and vWF expression levels but no significant correlation was found between growth patterns and cellular atypia or CD31 and vWF expression levels. Our study suggests that cellular atypia is useful for identifying differentiation levels in HSA cases. This study also provides useful information to determine differentiation levels of cell populations within HSA based only on morphological analysis, which will aid further HSA research such as identifying undifferentiation markers of endothelial cells or finding undifferentiated cell population in tissue sections.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently upregulated in prostate cancer, and its overexpression conveys tumor growth and angiogenesis by metabolizing asymmetric dimethylarginine (ADMA).

Tissue microarray analysis confirmed higher dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression in prostate cancer (PCa) compared to benign and normal prostate tissues. DDAH1 regulates nitric oxide (NO) production by degrading endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA). This study examined whether DDAH1 has any physiological role in PCa progression. Using overexpression of DDAH1 in PCa (PC3 and LNCaP) cell lines, we found that DDAH1 promotes cell proliferation, migration and invasion by lowering ADMA levels, as well as increasing NO production. VEGF, HIF-1α and iNOS were upregulated in DDAH1 expressing cells as result of elevated NO. DDAH1 increased secretion of pro-angiogenic signals bFGF and IL-8, into conditioned media. Treatment of DDAH1-positive PCa cells with NOS inhibitors (L-NAME and 1400 W) attenuated DDAH1 activity to promote cell growth. Xenografts derived from these cells grew significantly faster (> twofold) than those derived from control cells. Proliferation rate of cells stably expressing mutant DDAH1 was same as control cells unlike wild-type DDAH1-positive PCa cells. Xenograft tumors derived from mutant-positive cells did not differ from control tumors. VEGF, HIF-1α and iNOS expression did not differ in DDAH1 mutant-positive tumors compared to control tumors, but was upregulated in wild-type DDAH1 overexpressing tumors. Furthermore, CD31 immunostaining on xenograft tissues demonstrated that DDAH1 tumors had high endothelial content than mutant DDAH1 tumors. These data suggest that DDAH1 is an important mediator of PCa progression and NO/DDAH pathway needs to be considered in developing therapeutic strategies targeted at PCa.

High dengue virus load differentially modulates human microvascular endothelial barrier function during early infection.

Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.

New insights into the cellular makeup and progenitor potential of palatal connective tissues.

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco-gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de-epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de-epithelialised MSCs (deMSCs) and re-entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony-forming unit- fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA-DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA-, CD31-) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,-in the pericapillary regions and in remote regions of the lamina propria- and pericytes-surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision-making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

The influence of anti-TNF therapy on CD31 and VEGF expression in colonic mucosa of Crohn's disease patients in relation to mucosal healing.

INTRODUCTION: Immune-mediated angiogenesis may play an important role in the pathogenesis of inflammatory lesions in Crohn's disease (CD). The study aimed to assess the influence of anti-tumour necrosis factor (anti-TNF) therapy on the angiogenesis in relation to microscopic and endoscopic healing in CD patients. MATERIAL AND METHODS: Colonic tissue samples from 17 CD patients were taken during colonoscopy before and after anti-TNF therapy. Endoscopic and microscopic severities were estimated using validated scores. Immunohistochemical expression of CD31 and vascular endothelial growth factor (VEGF) were assessed in parallel. RESULTS: The expression of CD31 and VEGF decreased significantly after the anti-TNF therapy in parallel to endoscopic improvement; however, the microscopic activity did not change significantly. There was a correlation between the change in CD31 and VEGF expression (p = 0.01; r = 0.6), as well as endoscopic healing (p = 0.04; r = 0.4). CD31 immunoexpression correlated with the number of poly- and mononuclear cells in the infiltrates in the mucosal lamina propria before the therapy (p = 0.02; r = 0.5). CONCLUSIONS: We suggest that modulation of vascular proliferation can be a novel option to increase the efficacy of biological therapy in CD.

The adhesion molecule PECAM-1 enhances the TGF-ß-mediated inhibition of T cell function.

Transforming growth factor-ß (TGF-ß) is an immunosuppressive cytokine that inhibits the proinflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical TGF-ß signaling results in the activation of Smad proteins, which are transcription factors that regulate target gene expression. We found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitated noncanonical (Smad-independent) TGF-ß signaling in T cells. Subcutaneously injected tumor cells that are dependent on TGF-ß-mediated suppression of immunity for growth grew more slowly in PECAM-1(-/-) mice than in their wild-type counterparts. T cells isolated from PECAM-1(-/-) mice demonstrated relative insensitivity to the TGF-ß-dependent inhibition of interferon-γ (IFN-γ) production, granzyme B synthesis, and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-ß in a manner that was partially restored by reexpression of PECAM-1. Co-incubation of T cells with TGF-ß and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP-2). Such conditions also induced the colocalization of PECAM-1 with the TGF-ß receptor complex as identified by coimmunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-ß in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-ß inhibitory axis represents a means to overcome TGF-ß-dependent immunosuppression within the tumor microenvironment.

Localized signals that regulate transendothelial migration.

Transendothelial migration (TEM) of leukocytes is the step in leukocyte emigration in which the leukocyte actually leaves the blood vessel to carry out its role in the inflammatory response. It is therefore, arguably the most critical step in emigration. This review focuses on two of the many aspects of this process that have seen important recent developments. The adhesion molecules, PECAM (CD31) and CD99 that regulate two major steps in TEM, do so by regulating specific signals. PECAM initiates the signaling pathway responsible for the calcium flux that is required for TEM. Calcium enters through the cation channel TRPC6 and recruits the first wave of trafficking of membrane from the lateral border recycling compartment (LBRC). CD99 signals through soluble adenylate cyclase to activate protein kinase A to recruit a second wave of LBRC trafficking. Another process that is critical for TEM is transient removal of VE-cadherin from the site of TEM. However, the local signaling pathways that are responsible for this appear to be different from those that open the junctions to increase vascular permeability.

CD31 signals confer immune privilege to the vascular endothelium.

Constitutive resistance to cell death induced by inflammatory stimuli activating the extrinsic pathway of apoptosis is a key feature of vascular endothelial cells (ECs). Although this property is central to the maintenance of the endothelial barrier during inflammation, the molecular mechanisms of EC protection from cell-extrinsic, proapoptotic stimuli have not been investigated. We show that the Ig-family member CD31, which is expressed by endothelial but not epithelial cells, is necessary to prevent EC death induced by TNF-α and cytotoxic T lymphocytes in vitro. Combined quantitative RT-PCR array and biochemical analysis show that, upon the engagement of the TNF receptor with TNF-α on ECs, CD31 becomes activated and, in turn, counteracts the proapoptotic transcriptional program induced by TNF-α via activation of the Erk/Akt pathway. Specifically, Akt activation by CD31 signals prevents the localization of the forkhead transcription factor FoxO3 to the nucleus, thus inhibiting transcription of the proapoptotic genes CD95/Fas and caspase 7 and de-repressing the expression of the antiapoptotic gene cFlar. Both CD31 intracellular immunoreceptor tyrosine-based inhibition motifs are required for its prosurvival function. In vivo, CD31 gene transfer is sufficient to recapitulate the cytoprotective mechanisms in CD31(-) pancreatic ß cells, which become resistant to immune-mediated rejection when grafted in fully allogeneic recipients.

Characterization of CD133 Antibody-Directed Recellularized Heart Valves.

CD133mAb conjugation (CD133-C) hastens in vivo recellularization of decellularized porcine heart valve scaffolds when placed in the pulmonary position of sheep. We now characterize this early cellularization process 4 h, 3, 7, 14, 30, or 90 days post-implantation. Quantitative immunohistochemistry identified cell types as well as changes in cell markers and developmental cues. CD133(+)/CD31(-) cells adhered to the leaflet surface of CD133-C leaflets by 3 days and transitioned to native leaflet-like CD133(-)/CD31(+) cells by 30 days. Leaflet interstitium became increasingly populated with both alpha-smooth muscle actin (αSMA) and vimentin(+) cells from 14 to 90 days post-implantation. Wnt3a, and beta-catenin proteins were expressed at early (3-14 days) but not later (30-90 days) time points. In contrast, matrix metalloproteinase-2 and periostin proteins were increasingly expressed over 90 days. Thus, early development of CD133-C constructs includes a fairly rapid transition from a precursor cell adhesion/migration/transdifferentiation phenotype to a more mature cell/native valve-like matrix metabolism phenotype.