Dermal Microvessels Research Articles

Dimethylfumarate Inhibits Tumor-Necrosis-Factor-Induced CD62E Expression in an NF-κB-Dependent Manner

Fumaric acid esters are thought to improve psoriasis by altering leukocyte, keratinocyte, and/or endothelial functions. To determine specificity, kinetics, and molecular mechanisms of different fumaric acid esters in their ability to inhibit endothelial cell activation, we analyzed CD62E and CD54 expression in endothelial cells in vivo and in vitro. In lesional skin of psoriatic patients, oral fumaric acid ester treatment resulted in a marked reduction of CD62E but not CD54 expression on dermal microvessels. Using human umbilical vein endothelial cells, dimethylfumarate almost completely inhibited tumor-necrosis-factor-induced CD62E, but not CD54 expression at concentrations < or = 70 microM, mimicking the situation in vivo. A 60 min dimethylfumarate preincubation was sufficient to block tumor-necrosis-factor-induced CD62E expression for up to 24 h. In contrast, equimolar concentrations of methylhydrogenfumarate, the hydrolysis product of dimethylfumarate, did not suppress tumor-necrosis-factor-induced CD62E expression. Likewise, all fumaric acid esters other than dimethylfumarate were ineffective. Using CD62E, NF-kappa B, or AP-1-responsive promoter constructs, dimethylfumarate inhibited tumor-necrosis-factor-induced activation of the CD62E and the NF-kappa B but not the AP-1 promoter construct. In summary, at a dose range < or = 70 microM, dimethylfumarate appeared to be a specific inhibitor of CD62E expression in an NF-kappa B-dependent manner.

Decreased mRNA Stability as a Mechanism of Glucocorticoid-Mediated Inhibition of Vascular Endothelial Growth Factor Gene Expression by Cultured Keratinocytes

Epidermal keratinocyte-derived overexpression of vascular endothelial growth factor has been functionally linked to increased density of tortuous and hyperpermeable dermal microvessels, representing a characteristic component of cutaneous inflammation. We hypothesized that potent anti-inflammatory properties of synthetic glucocorticoids are attributed in part to their interference with the regulated vascular endothelial growth factor expression by keratinocytes. As vascular endothelial growth factor is markedly upregulated by autocrine transforming growth factor alpha and paracrine hepatocyte growth factor/scatter factor expression, the effect of glucocorticoids on growth-factor-induced vascular endothelial growth factor production by primary and immortalized keratinocytes was examined. Glucocorticoids were shown to suppress vascular endothelial growth factor protein and mRNA expression in a concentration- and time-dependent fashion. In transcriptional activation studies, however, common 5'-regulatory regions of the vascular endothelial growth factor gene failed to confer inhibitory glucocorticoid effects. Instead, glucocorticoids were shown to increase vascular endothelial growth factor mRNA turnover, indicating that post-transcriptional modes of glucocorticoid action are employed to negatively regulate induced vascular endothelial growth factor expression. Together, these studies identify vascular endothelial growth factor upregulation by epidermal keratinocytes as a putative target of glucocorticoid action in cutaneous inflammation. Our data provide strong evidence that mRNA destabilization may represent a mechanism by which glucocorticoids inhibit growth-factor-regulated vascular endothelial growth factor gene expression by keratinocytes.

Tamoxifen inhibits endothelial cell proliferation and attenuates VEGF-mediated angiogenesis and migration in vivo

Introduction: Angiogenesis is fundamental to tumour growth and vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic cytokines known. We have previously demonstrated that tamoxifen reduces serum VEGF in certain cancer patients. We hypothesized that tamoxifen may attenuate the angiogenetic response to VEGF. Methods: Human dermal microvessel endothelial primary cell cultures (HMEC) were incubated with tamoxifen (1.25–5.0 μg) or vehicle. Cell proliferation was quantified using 5-bromo-2′-deoxyuridine (BrdU) labelling endothelial cell proliferation assay. The effect of oral tamoxifen (20 mg/day) on VEGF-mediated angiogenesis in vivo was assessed using a Matrigel angiogenesis assay in the Sprague–Dawley rat. Results: Tamoxifen (5.0 μg/ml) significantly reduced HMEC proliferation over 24 h when compared with cells treated with vehicle alone. Oral administration of tamoxifen in the rat (20 mg/day) significantly reduced endothelial cell proliferation and migration in response to VEGF. Conclusion: Tamoxifen (5.0 μg/ml) reduces proliferation of a VEGF-dependent endothelial cell line in vitro. In vivo, orally administered tamoxifen reduces VEGF-mediated angiogenesis in the rat. These findings indicate that tamoxifen may directly inhibit the effect of VEGF on the endothelial cell, in addition to its previously described effect of reducing serum VEGF levels. This data supports a role for tamoxifen in modulation of the VEGF-dependent angiogenic response to surgical trauma, particularly as an adjuvant therapy for VEGF-dependent tumours.

Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide.

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.

Noncytolytic human lymphocytes injure dermal microvessels in the huPBL-SCID skin graft model

Recent transplantation experiments using perforin-deficient mice as allograft recipients have challenged the concept that allograft rejection is mediated exclusively by CTL. We sought to determine if human noncytolytic lymphocytes could mediate rejection of allogeneic human skin grafts in the huPBL-SCID mouse model of rejection. We generated short term lines of human lymphocytes from peripheral blood mononuclear cells using PHA as a mitogen. The first group was stimulated with PHA alone, the second with PHA plus IL-4 and neutralizing antibody to IL-12, and in the third group PBL were depleted of B cells and monocytes before stimulation as in group 2. After two passages, lines were tested for cytolytic ability and IFN-γ production. Each line was injected i.p. to mice bearing allogeneic skin grafts. The grafts were harvested between day 16 and 21 after PBL injection, then the histology was scored by a blinded observer for degree of infiltration, microvessel injury, induction of epidermal MHC class II, and perforin expression. In vitro we found that PBL in groups 2 and 3 were unable to lyse cultured endothelial cells in a lectin-directed 111In release assay. In vivo 80% of the IL-4/anti-IL-12 groups maintained the IFN-γ-low phenotype, and no perforin was detected in these grafts. Nevertheless, human microvessel injury was similar between the two groups. This was not antibody-dependent since the B-cell-depleted group showed similar injury. Moreover adjacent murine vessels were intact. We interpret these observations to show (1) these human PBL lines maintained their phenotype following in vivo restimulation, and (2) noncytolytic graft-infiltrating lymphocytes specifically promote injury of allogeneic human microvessels.

Isolation and in Vitro Characterization of Human Dermal Microvascular Pericytes

Pericytes cover the abluminal surface of capillaries and venules and are thought to play an important role in microvascular regulation and pathology. The purpose of this study was to isolate and characterize human dermal microvascular pericytes (HDMPC), a minor cell type in the skin but a relatively easily obtainable human source of tissue. We developed and compared two procedures that differed in the preselection method. Isolation of dermal microvessel fragments from neonatal foreskins by trypsin digestion was followed by mechanical release of subepidermal tissue, collagenase treatment, and sieving through 100- and 30-μm meshes. After subcultivation, pericytes were preselected either by isolation of outgrowing capillary fragments or by 3G5-coupled magnetic beads. Pericytes were selected finally by cultivation of single cells in endothelial cell-conditioned media. Cultured HDMPC were seen to be large and well spread with irregular edges and prominent stress fibers. They lack contact inhibition, are positive for 3G5 antigen, α-smooth muscle actin, and vimentin, and are negative for the endothelial cell marker CD31, diI-acetylated low-density lipoprotein uptake, cytokeratin 5, 6, and 18, and S100 protein. Using both preselection methods, we could establish purified cell cultures of HDMPC. The results of these studies represent the first report of HDMPC isolation.

Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4.

In HIV-infected patients, concurrent infections with bacteria and viruses are known to induce HIV replication as assessed by increases in plasma HIV RNA levels. In the present study, we determined the cell surface receptor and molecular mechanisms of enterobacterial LPS-induced HIV transcription. Human dermal microvessel endothelial cells (HMEC) were transfected with an HIV-long terminal repeat (LTR)-luciferase construct and subsequently stimulated with purified bacterial LPS. Our studies demonstrate that human Toll-like receptor 4 (TLR4) mediates LPS-induced NF-kappaB and HIV-LTR activation in HMEC through IL-1 signaling molecules, namely myeloid differentiation protein, IL-1R-associated kinase, TNFR-associated factor, and NF-kappaB-inducing kinase. Cotransfection of HMEC with HIV-LTR-luciferase and TLR4 cDNA from LPS-hyporesponsive C3H/HeJ mice abrogates LPS-induced HIV transcription as does the use of dominant-negative mutants of the IL-1 signaling molecules. Transfection of HMEC with an HIV-LTR-mutant that lacks the NF-kappaB binding site or pretreatment of cells with chemical inhibitors of the NF-kappaB pathway also blocked LPS-induced HIV-LTR transactivation. These data support the conclusion that TLR4 mediates enterobacterial LPS-induced HIV transcription via IL-1 signaling molecules and NF-kappaB activation plays an important role in HIV-LTR transactivation.

Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation.

Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-alpha, or IFN-gamma induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-gamma act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-kappaB dependent. LPS and IFN-gamma also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-alpha, or IFN-gamma. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-gamma, in inducing shock associated with Gram-negative sepsis.

Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells

P29 Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.

Specialized Contributions by α(1,3)-Fucosyltransferase-IV and FucT-VII during Leukocyte Rolling in Dermal Microvessels

Noninflamed skin venules support constitutive leukocyte rolling. P-selectin controls the rolling frequency, whereas E-selectin dictates rolling velocity (Vroll). Fucosylated selectin ligands are essential for all interactions, as rolling was absent in mice doubly deficient in α1,3-fucosyltransferase (FucT)-IV and FucT-VII. The rolling fraction was reduced in FucT-VII−/− animals but normal in FucT-IV−/− mice. However, Vroll was markedly increased in both strains. P-selectin ligands generated by FucT-VII are crucial for initial leukocyte tethering, whereas E-selectin ligands that permit maximum slowing of Vroll require simultaneous expression of FucT-IV and FucT-VII. These results demonstrate a role for FucT-IV in selectin-dependent adhesion and suggest that the endothelial selectins and FucTs have distinct but overlapping functions in the immunosurveillance of the skin.

Hepatocyte Growth Factor Enhances MMP Activity in Human Endothelial Cells

Scatter factor (SF) or hepatocyte growth factor (HGF) has been identified as an angiogenic factor. Angiogenesis requires not only tube formation but also invasion of pericytes and extracellular matrix (ECM) remodeling to promote new vessel stabilization. In the current study, the effect of SF/HGF on endothelial cell (EC) production of matrix metalloproteinases (MMPs) was explored. We showed that SF/HGF enhanced MT1-MMP synthesis and induced MMP-2 activation in two human EC lines: dermal microvessel EC and coronary arterial EC. Furthermore, SF/HGF accelerated EC invasion into matrix, an activity that could be inhibited by a MMP inhibitor. We also demonstrated that the MAP kinase cascade is critical in signal transduction pathway from SF/HGF stimulation to MT1-MMP up-regulation. The current study indicates that MMP activation is a novel effect of SF/HGF on ECs.

Bacterial Lipopolysaccharide Activates NF-κB through Toll-like Receptor 4 (TLR-4) in Cultured Human Dermal Endothelial Cells: DIFFERENTIAL EXPRESSION OF TLR-4 AND TLR-2 IN ENDOTHELIAL CELLS

A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non-responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal.

Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ.

Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.

Neutrophil inhibitory factor abrogates neutrophil adhesion by blockade of CD11a and CD11b beta(2) integrins.

We studied the basis of inhibition of polymorphonuclear leukocyte (PMN) adhesion induced by neutrophil inhibitory factor (NIF), a 41-kDa CD11/CD18 beta(2) integrin-binding protein isolated from the canine hookworm (Ancylostoma caninum). NIF blocked PMN adhesion in a concentration-dependent manner with complete blockade occurring at approximately 10 nM NIF. Because CD11a and CD11b beta(2) integrins are functionally active on stimulated PMNs, and yet NIF is postulated to inhibit only CD11b integrin by binding to its I domain, we evaluated the contributions of CD11a and CD11b beta(2) integrins in the mechanism of inhibition of PMN adhesion to endothelial cells. We observed an additive inhibitory effect (>90% inhibition) of PMN adhesion to endothelial cells when NIF was used in combination with anti-CD11b monoclonal antibodies, which alone at saturating concentrations reduced PMN adhesion by only 50%. NIF also prevented aggregation of phorbol ester-stimulated JY lymphoblastoid cells that expressed only the functionally active CD11a, suggesting that NIF also can inhibit CD11a-dependent response. We transduced the NIF cDNA into human dermal microvessel endothelial cells in which NIF synthesis and release prevented PMN adhesion to the transduced human dermal microvessel endothelial cells. These data indicated that the potent antiadhesive effect of NIF may be the result of inhibition of CD11a and CD11b beta(2) integrins on PMNs. Moreover, the strategy of NIF release from transduced endothelial cells suggests the feasibility of blocking the CD11a- and CD11b beta(2) integrin-dependent PMN adhesion and PMN migration responses specifically at sites of endothelial cell activation.

Bacterial Lipopolysaccharide Activates Nuclear Factor-κB through Interleukin-1 Signaling Mediators in Cultured Human Dermal Endothelial Cells and Mononuclear Phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.

Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice.

Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.

Relationship between telomerase activity, proliferative activity and apoptosis in placenta of normal and abnormal prengancy

Tissue hypoxia is a characteristic feature of malignant tumors and healing wounds, conditions that are associated with angiogenesis and with increased expression of vascular permeability factor (VPF; also called vascular endothelial growth factor, VEGF), a selective endothelial cell mitogen inducing microvascular hyperpermeability in vivo. We investigated the regulation of VPF/VEGF and its receptors by tissue hypoxia in normal human skin explants and in cultured skin cells in vitro. VPF/VEGF mRNA expression was dramatically upregulated in epidermal keratinocytes, dermal fihroblasts, and dermal microvessels after 24 h of skin organ culture. Hypoxia also enhanced the expression of VPF/VEGF in cultured epidermal keratinocytes and dermal microvascular endothelial cells (predominantly VPF/VEGF121 and V-PF/VEGF165) and in dermal fibroblasts (additional upregulation of VPF/VEGF189). The expression of the VPF/VEGF receptor Fit-1 was selectively induced on dermal microvessels in skin explant cultures and in dermal endothelial cell monolayer cultures under hypoxic conditions. In contrast, the KDR receptor was downregulated by hypoxia. These results suggest that hypoxia likely regulates cutaneous angiogenesis and mierovascular permeability by two distinct mechanisms: (i) Induction of VPF/VEGF in epithelial and mesenchymal cells, including endothelial cells. (ii) Differential modulation of VPF/VEGF receptor expression by microvascular endothelial cells. These mechanisms may be of importance in the pathogenesis of healing wounds and some malignant tumors that are commonly characterized by hypoxia and overexpression of VPF/VEGF.

A microangiographic technique using synchrotron radiation to visualize dermal circulation in vivo.

Conventional angiography cannot resolve dermal small vessels with a diameter of 200 microm or less. In vitro microangiography is currently characterized by better spatial resolution than conventional angiography but does not allow visualization of the blood stream in dermal vessels in vivo. In this study, we introduce a novel synchrotron radiation microangiographic system for visualizing the structure of and blood flow in dermal microvessels in vivo repeatedly. We used monochromatic synchrotron radiation with an energy just above the k-edge of iodine (33.3 keV) as an x-ray and a high-definition television camera system with a high-sensitivity image pick-up tube for detection. The 33.3-keV monochromatic synchrotron radiation allows detection of a small amount of iodine, and the high-definition television camera system can resolve small vessels with high-spatial resolution and no loss of sensitivity. We performed synchrotron radiation angiography of superficial inferior epigastric arteries and their branches in nine rats, and of the caudal artery in 14 rats, and compared angiographic images taken by the current system with those taken by a conventional angiographic system in seven rats. With this new microangiographic technique, we could visualize small dermal vessels with a diameter as low as 50 microm. In addition, repeated angiograms at baseline and under increasing body temperature could be obtained. This new microangiographic approach is expected to be very useful for the assessment of dermal circulation in patients.

Dermal dendrocytes participate in the cellular pathology of experimental acute graft-versus-host disease.

In a well established murine model relevant to human disease, graft-versus-host disease results from recognition of recipient minor histocompatibility antigens by donor bone marrow-derived T lymphocytes. Previous studies suggest that factor XIIIa-positive dermal dendrocytes may be involved in the pathogenesis of disorders involving antigen presentation to T cells and dermal fibrosis. This study was undertaken to determine (i) whether normal murine skin contains factor XIIIa-positive dermal dendrocytes, and (ii) whether such cells participate in the pathophysiology of acute graft-versus-host disease. Graft-versus-host disease was produced using B10.BR CD8+ donor T cells administered to CBA recipients. Skin samples were collected weekly for a 5-week period and evaluated by immunohistochemistry and electron microscopy. Our data indicate that normal murine dermis contains factor XIIIa-positive cells localized primarily around deep dermal microvessels. Ultrastructural analyses reveal these cells to have long processes, pinocytotic vesicles, fibronexuses, and intimate associations with mast cells. During graft-versus-host disease, factor XIIIa-positive dendrocytes appeared within the superficial dermis. By ultrastructure, the dendrocytes were hypertrophic and highly branched, and demonstrated an intimate relationship with neighboring cells. In conclusion, factor XIIIa-positive dendrocytes comprise a normal component of the murine dermis and undergo alterations in experimental acute graft-versus-host disease consistent with participation in disease pathophysiology.