Differential endothelial cell gene expression by African Americans versus Caucasian Americans: a possible contribution to health disparity in vascular disease and cancer.

BACKGROUND: Health disparities and the high prevalence of cardiovascular disease continue to be perplexing worldwide health challenges. This study addresses the possibility that genetic differences affecting the biology of the vascular endothelium could be a factor contributing to the increased burden of cardiovascular disease and cancer among African Americans (AA) compared to Caucasian Americans (CA). METHODS: From self-identified, healthy, 20 to 29-year-old AA (n = 21) and CA (n = 17), we established cultures of blood outgrowth endothelial cells (BOEC) and applied microarray profiling. BOEC have never been exposed to in vivo influences, and their gene expression reflects culture conditions (meticulously controlled) and donor genetics. Significance Analysis of Microarray identified differential expression of single genes. Gene Set Enrichment Analysis examined expression of pre-determined gene sets that survey nine biological systems relevant to endothelial biology. RESULTS: At the highly stringent threshold of False Discovery Rate (FDR) = 0, 31 single genes were differentially expressed in AA. PSPH exhibited the greatest fold-change (AA > CA), but this was entirely accounted for by a homolog (PSPHL) hidden within the PSPH probe set. Among other significantly different genes were: for AA > CA, SOS1, AMFR, FGFR3; and for AA < CA, ARVCF, BIN3, EIF4B. Many more (221 transcripts for 204 genes) were differentially expressed at the less stringent threshold of FDR <.05. Using the biological systems approach, we identified shear response biology as being significantly different for AA versus CA, showing an apparent tonic increase of expression (AA > CA) for 46/157 genes within that system. CONCLUSIONS: Many of the genes implicated here have substantial roles in endothelial biology. Shear stress response, a critical regulator of endothelial function and vascular homeostasis, may be different between AA and CA. These results potentially have direct implications for the role of endothelial cells in vascular disease (hypertension, stroke) and cancer (via angiogenesis). Also, they are consistent with our over-arching hypothesis that genetic influences stemming from ancestral continent-of-origin could impact upon endothelial cell biology and thereby contribute to disparity of vascular-related disease burden among AA. The method used here could be productively employed to bridge the gap between information from structural genomics (for example, disease association) and cell function and pathophysiology.

Identification and functional assessment of endothelial P1H12.

Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca(++)-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO(P1H12))-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-kappa B to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.

Thrombin-stimulated calcium mobilization is inhibited by thrombospondin via CD36.

Activation of the G-protein-linked thrombin receptor in endothelial cells normally leads to an increase in free intracellular calcium, [Ca2+]i, which is the proximate stimulus for many important cell functions. We present evidence showing that signals from CD36, the thrombospondin (TSP) receptor, can inhibit this thrombin-mediated calcium response. Human endothelial cells preloaded with Indo-1 exhibited rapid calcium mobilization in response to thrombin. The presence of TSP inhibited the thrombin-stimulated calcium response in CD36-positive microvascular endothelial cells but not in CD36-negative umbilical vein endothelial cells. This TSP effect was mimicked by anti-CD36 antibodies and a TSP peptide (CSVTCG), but not by an alternative CD36 ligand (collagen IV) or an antibody to an alternative TSP receptor (alphavbeta3). TSP also inhibited the calcium response to the thrombin receptor-tethered ligand peptide, SFLLRN. In addition, TSP and anti-CD36 antibodies inhibited the calcium response of a closely related receptor, the trypsin/SLIGKVD-activated receptor PAR-2. TSP did not indiscriminately inhibit all calcium release pathways, since histamine- or VEGF-stimulated calcium responses were not inhibited by TSP. We conclude that cross-talk from the CD36 receptor influences the responsive state of the endothelial thrombin receptor family and/or its signaling pathway.

Confocal microscopic analysis of integrin expression on the microvasculature and its sprouts in the neonatal foreskin.

Members of the integrin family of adhesion receptors are essential participants in blood vessel growth and remodeling. It is not known which integrins are involved in the initial stages of angiogenesis in vivo. In this study we determined the location of integrins on the blood vessels of a growing tissue, the neonatal foreskin, in which neovascularization is likely to occur. We used the confocal microscope to visually reconstruct vessels from the papillary dermis of the foreskin and to identify potential sprouts as narrow, tapering extensions from these vessels. Blood vessels were initially identified by their positive reaction with antibodies to von Willebrand factor or human platelet endothelial cell adhesion molecule and their negative response to anti-neurofilament antibodies. Later, vessels were identified by their shape and location. We screened vessels with anti bodies to integrin subunits alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1, beta 3 and beta 4. We found that integrin subunits alpha 6 and beta 4 were consistently found along the whole length of capillary loops and extended to the distal ends of presumed sprouts. The alpha 2 and alpha v integrin concentrations, which are normally low in the microvasculature, were increased on the sprouts. alpha 5 was either absent from vessels entirely or more concentrated on the body than on the sprout. alpha 1 was more commonly present on nerves than blood vessels. These studies suggest an important role for the alpha 6 beta 4 integrin in the initial stages of endothelial outmigration during new vessel growth.

Basic FGF and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells.

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) are known to alter the migratory and proliferative capacity of endothelial cells in vitro and to stimulate angiogenesis in vivo. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor expression and activity. We examined the ability of these growth factors to modulate the expression of specific integrins in human microvascular endothelial cells (MEC). Immunoprecipitation of metabolically labeled MEC showed that bFGF upregulated the biosynthesis of alpha 2, alpha 5, beta 1, and beta 3. bFGF induced an increase in the levels of mRNA for alpha 2 and beta 1. TGF-beta increased synthesis of alpha 2, alpha 5, and beta 1. These results suggest that bFGF and TGF-beta selectively alter integrin profiles and influence interactions of MEC with the extracellular matrix during neovascularization. In particular, the upregulation of the collagen/laminin receptor, alpha 2 beta 1, by bFGF may provide activated endothelial cells with an enhanced capacity to migrate through both their underlying basement membrane and the interstitial matrix.

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.

Epithelial and neural localization and heparin binding of the cell-substratum adhesion molecule, epinectin.

Epinectin, a cell-substratum adhesion promoting molecule, was first isolated from the extracellular matrix of A431 human squamous carcinoma cells. In order to determine the biologic significance of epinectin, we determined the distribution of epinectin in various rat epithelial tissues by indirect immunofluorescence microscopy. Polyclonal antibodies to epinectin stained basal cells and basilar regions of skin, urinary bladder, and vagina. There was predominantly cytoplasmic staining along with amorphous extracellular staining. Strong staining was also noted in sebaceous glands and hair follicles. The immunoreactivity for epinectin in the skin was distinct from that for fibronectin, laminin, and type IV collagen. Antibodies to epinectin also stained subpopulations of neurons in the cerebrum and cerebellum. Epinectin antibodies strongly stained the cytoplasm of some pineal cells and cells of the pars intermedia of the pituitary. The distribution of epinectin suggests a role not only in epithelial cell-substratum adhesion, but in neuronal cell function. Heparan sulfate is known to be involved in the binding of several adhesion promoting molecules to cell surfaces. In order to assess the mechanism of adhesion of epinectin to cells, we measured the binding of 3H-heparin to epinectin. Binding of 3H-heparin was concentration dependent and inhibitable with cold heparin.

Isolation and characterization of epinectin, a novel adhesion protein for epithelial cells.

A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K., M. Kurkinen, K. Alitalo, A. Vaheri, S. Johansson, and M. Höök, 1979 J. Cell Biol., 81:83-91) and Yamada and Weston (Yamada, K., and J. A. Weston, 1974, Proc. Natl. Acad. Sci. USA, 71:3492-3496). Cells were solubilized with 0.5% deoxycholate, 10 mM Tris, 0.9% NaCl, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. The residual matrix was then removed from the plates with 6 M urea and 1 mM phenylmethylsulfonyl fluoride and phosphate-buffered saline. SDS PAGE gels of the 6 M urea extract showed one major band at 70,000-mol-wt by Coomassie Blue staining. A 70,000-mol-wt isotopically-labeled band could also be extracted from the matrix of cells incubated with [35S]methionine. Because of the presence of this protein on squamous-derived epithelial cells we have called the 70,000-mol-wt molecule epinectin. Indirect immunofluorescence with polyclonal rabbit antibodies against epinectin stained A431 cells pericellularly in dense punctate accumulations and along the plasma membrane. Enzyme-linked immunoassays and gel-transfer immunolocalization studies showed that the extract did not cross-react with antibodies to fibronectin, laminin, serum-spreading factor, epibolin, or keratin. Additionally, antibodies to epinectin did not cross-react with these proteins. Further studies showed that epinectin does not bind to gelatin. Cell-adhesion assay, using radiolabeled A431 carcinoma cells on various adhesion-promoting substrates, showed that epinectin has similar adhesion-promoting capacity as serum-spreading factor, was somewhat less active than fibronectin, but more effective than laminin or epibolin. Epinectin appears to be a unique protein isolated from epidermoid tumor cells that is distinct from other known adhesion proteins.

Peptide fragments of laminin and fibronectin promote migration (haptotaxis and chemotaxis) of metastatic cells.

The migration of tumour cells through basement membranes and extracellular matrices is an integral component of tumour invasion and metastasis. Laminin (LMN) and fibronectin (FN) at 1-100 micrograms/ml promote the directed migration of metastatic murine melanoma cells 40-70-fold greater than controls in modified Boyden chambers. Antibodies abrogated the migration of cells in response to the respective protein. Preincubation of melanoma cells with plasma FN had no effect on subsequent migration to LMN or FN. The migration of these cells was largely related to substratum-attached molecules and increasing adhesion gradients of cells; this has been termed haptotaxis. Peptide fragments of both FN and LMN were isolated by affinity chromatography with monoclonal antibodies, heparin or other constituents. FN has two unique domains, 80-125 K and 66 K, which promote the adhesion of tumour cells, whereas only one appeared to be responsible for promoting migration. Peptides of LMN, isolated with heparin and monoclonal antibody, define a cell migration-promoting activity within the 200 K chains of LMN. Serum spreading factor and epinectin, the latter an adhesion molecule derived from squamous epithelial tumour cells, are also capable of promoting the migration of malignant cells. Thus, directed migration of metastatic tumour cells may be promoted with peptide fragments of adhesion molecules and blocked with the respective antibody.