Expression and function of endothelial cell alpha v integrin receptors in wound-induced human angiogenesis in human skin/SCID mice chimeras.

Accumulating evidence indicates that endothelial cell integrins that bind to the matrix proteins associated with inflammation and wound healing are involved in the process of angiogenesis. The integrins containing the alpha v subunit appear to be particularly important. To study the involvement of these receptors in human angiogenesis, a model of wound-associated human angiogenesis was established in human skin transplanted onto severe combined immunodeficient (SCID) mice. Using this model, we studied the expression of several alpha v integrins and tested the hypothesis that blockage of the alpha v beta 3 integrin would inhibit human angiogenesis during human wound healing. These studies revealed that the alpha v beta 3, alpha v beta 5, and alpha v beta 6 integrins are up-regulated briefly during wound angiogenesis with different patterns of expression and that inhibition of the alpha v beta 3 integrin blocked new vessel formation during human wound healing.     

Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair.     

Distribution of the alpha 1-alpha 6 integrin subunits in human developing and term placenta

The distribution of the alpha 1-alpha 6 as well as alpha v, beta 1, beta 3 and beta 4 integrin subunits in human first and second trimester and term placentas was studied by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (mAbs). In first and second trimester villi, the alpha 1 and beta 1 integrin subunits were detected in the stromal cells, that were mostly also immunoreactive for desmin. Desmin-positive stromal cells were also found in villi of term placentas, but the stroma was negative for anti-alpha 1 and -beta 1. In the villous trophoblast, anti-alpha 6 and -beta 4 revealed a distinct basal immunoreactivity during all stages of development, whereas immunoreactivity for the alpha 3 and beta 1 subunits emerged during the second and third trimesters. Throughout placental development, endothelia of villous capillaries reacted prominently with anti-alpha 1 and -beta 1. Intermediate trophoblastic cells displayed a somewhat heterogenous immunoreactivity for the beta 1, alpha 1, alpha 3 and alpha 5 integrin subunits, and differed from villous trophoblast also in their lack of expression of the alpha 6 and beta 4 subunits. While nondecidualized endometrial cells displayed weak reactivity for the alpha 1 and beta 1 integrin subunits, the individual decidual cells presented both a basement membrane and a cell surface-confined immunoreactivity for anti-alpha 1, -alpha 3, and -beta 1. The results suggest a role for integrins in placental development, and show that expression of integrins is modulated during the differentiation of trophoblast, villous stroma, and decidual cells. Furthermore, the basal localization of alpha 6 beta 4 and alpha 3 beta 1 integrins suggests that they are employed as basement membrane receptors in the villous trophoblast, and the emergence of the alpha 3 beta 1 complex may reflect that the cytotrophoblast and syncytiotrophoblast recognize the basement membrane differently.     

Integrin characterization in pulmonary bronchioles

Integrins are a family of cell surface glycoproteins that act as receptors for ECM proteins or for membrane bound counter-receptors on other cells. The integrin receptor family of vertebrates includes at least 16 distinct alpha subunits and at least 8 beta subunits which can associate to form more than 20 distinct integrins. So far, there are no published reports describing integrin characterization in mouse lung tissue and mouse Clara cells. This paper described the characterization of six integrins, mainly alpha(5), alpha(v), alpha(6), beta(1), beta(3), and beta(4), in mouse pulmonary bronchioles and also in Clara cell cultures. alpha(5), alpha(v), alpha(6), beta(1), and beta(4) integrins were present in Clara cells both in tissue sections and cultures. beta(3) integrin was found to be absent in mouse Clara cells.     

RGD-dependent vacuolation and lumen formation observed during endothelial cell morphogenesis in three-dimensional fibrin matrices involves the alpha(v)beta(3) and alpha(5)beta(1) integrins

Recent data have revealed the involvement of the alpha(v)beta(3) integrin in angiogenesis. However, few studies to date have provided a convincing role for this receptor in in vitro assays of endothelial cell morphogenesis where defined steps can be examined. Here, we present data showing that two integrins, alpha(v)beta(3) and alpha(5)beta(1), regulate human endothelial cell vacuolation and lumen formation in three-dimensional fibrin matrices. Cells resuspended in fibrin formed intracellular vacuoles that coalesced into lumenal structures. These morphogenic events were completely inhibited by the simultaneous addition of anti-alpha(v)beta(3) and anti-alpha(5) integrin antibodies. Complete blockade was also accomplished with a combination of the cyclic Arg-Gly-Asp (cRGD) peptide and anti-alpha(5) integrin antibodies. No blockade was observed with the control Arg-Gly-Glu (RGE) peptide alone or in combination with control antibodies. Finally, we were able to demonstrate regression of vacuoles and lumens several hours after the addition of cRGD peptides combined with anti-alpha(5) integrin antibodies. These effects were not observed with control peptides alone or in combination with control antibodies. We report here the novel involvement of both the alpha(v)beta(3) and alpha(5)beta(1) integrins in vacuolation and lumen formation in a fibrin matrix, implicating a role for multiple integrins in endothelial cell morphogenesis.     

Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.     

Short-term effects of adhesion peptides on the responses of preosteoblasts to pBMP-9

Adhesion peptides are currently used to enhance the interactions of osteoblasts with biomaterials. However, little is known about the effects of adhesion peptides on cell responses to growth factors, especially the bone morphogenetic proteins (BMPs). We used adhesion peptides Ac-CGGNGERPRGDTYRAY-NH(2) (pRGD), derived from bone sialoprotein, and Ac-CGGDGEA-NH(2) (pDGEA), derived from collagen, which interact with alpha(v)beta(3) and alpha(2)beta(1) integrins, respectively. We analyzed the effects of pRGD- and pDGEA-coated polystyrene (PS) on the responses of murine MC3T3-E1 preosteoblasts to a peptide derived from human BMP-9 (pBMP-9) in serum-free medium. After 1h, pRGD favoured interactions with alpha(v) while pDGEA bound beta(1) integrin subunits. Adding pBMP-9 (400 ng/mL) increased the amount of alpha(v) integrin subunits in cell membranes on pRGD-coated PS, but had no effect on beta(1) integrin subunits. Only on this substratum, collagen type I mRNA was enhanced and the addition of pBMP-9 promoted the early cell differentiation, increasing their alkaline phosphatase (ALP) activity within 24 h. These cells also organized beta(1) integrin subunits at their focal adhesion points. Inhibiting alpha(2)beta(1) integrins by pDGEA pre-treatment decreased this ALP activity. It is therefore important to understand the impact of adhesion peptides on the early cell responses to growth factors in order to improve biomimetic materials.     

Anti-inflammatory effects of alphav integrin antagonism in acute kidney allograft rejection

Integrin-mediated cell adhesion and signaling is essential to vascular development and inflammatory processes. Elevated expression of integrin alpha(v)beta(3) has been detected in ischemia-reperfusion injury and rejecting heart allografts. We thus hypothesized that the inhibition of alpha(v)-associated integrins may have potent anti-inflammatory effects in acute kidney allograft rejection. We studied the effects of a peptidomimetic antagonist of alpha(v) integrins in two rat models of renal allotransplantation, differing in degree of major histocompatibility complex mismatch. Integrin alpha(v)beta(3) was up-regulated in rejecting renal allografts. Integrin antagonist reduced the histological signs of acute rejection, the intensity of the mononuclear cell infiltration, and cell proliferation in the grafted kidneys. This could be correlated to a reduced leukocyte-endothelial interaction and an improved peritubular microcirculation observed by intravital microscopy. In vitro under laminar flow conditions, the arrest of monocytes to interleukin-1beta-activated endothelium was decreased. Furthermore, in co-culture models the proliferation and transmigration of monocytes/macrophages, endothelium, and fibroblasts induced by renal tubular epithelia was efficiently inhibited by alpha(v) integrin antagonism. These data reveal an important role of this integrin subclass in leukocyte recruitment and development and maintenance of acute rejection; blockade of alpha(v) integrins may provide a new therapeutic strategy to attenuate acute allograft rejection.     

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.     

Amino acid residues required for binding of vascular cell adhesion molecule-1 to integrin alpha 4 beta 7

The homologous Ig-like domains 1 and 4 of vascular cell adhesion molecule (VCAM)-1 present binding sites to the leukocyte integrins alpha 4 beta 1 and alpha 4 beta 7 . In the present study, amino acid substitution mutants were used to identify sequence motifs mediating binding of integrin alpha 4 beta 7 to the first domain of VCAM-1. We demonstrate that binding of integrin alpha 4 beta 7 to VCAM-1 containing the D40A mutation located in the loop between beta strands C1 and D1 was completely abrogated and was not restored by activating integrin binding functions with Mn2+. Thus, the I(39)DSP motif functions as a central recognition site for integrin alpha 4 beta 7. Analysis of the E66A mutation demonstrated that the G(64)NEH sequence, which is exposed on the loop structure between beta strands E1 and F1, represents an additional recognition site for alpha 4 beta 7 integrin. However, the inhibitory effect of the E66A mutation on cell binding was not specific for alpha 4 beta 7 but was also observed for integrin alpha 4 beta 1. In contrast to the I(39)DSP and G(64)NEH sequences, the K(79)LEK motif present in beta strand G1 was involved in binding to alpha 4 beta 1 but not alpha 4 beta 7. The function of G(64)NEH and K(79)LEK motifs in alpha 4++-integrin interactions was confirmed by divalent cation titration assays and peptide inhibition studies. Integrin binding to E66A or E81A;K82A mutants was restored by activation with saturating concentrations of Mn2+. Binding of both alpha 4 beta 1 and alpha 4 beta 7 integrins was not affected by E29A, R36A, E50A or E87A mutations. Together, these results identify the I(39)DSP and G(64)NEH motifs as common recognition sites for both alpha 4 beta 1 and alpha 4 beta 7 integrins, whereas the K(79)LEK sequence appears to confer specificity for alpha 4 beta 1 binding.      

The effect of gonadotrophic stimulation on integrin expression in the endometrium.

BACKGROUND: Despite many recent advances in IVF treatment implantation rates per embryo transfer rarely exceed 30%. Three integrins (alpha(1)beta(1),alpha(4)beta(1) and alpha(v)beta(3)) have been shown to be expressed in the endometrium in a cyclically dependent manner and are thought therefore to play a vital role in the process of implantation. METHODS: The effect of gonadotrophin stimulation on the expression of these three integrins within the endometrium was investigated by examining biopsies from oocyte donation patients and comparing them with fertile controls. RESULTS: A delay in the maturation of the glandular epithelium was found in the oocyte donation patients. There was also a reduction in the expression of all three integrins in the glandular epithelium and also a reduced expression of the alpha(v)beta(3) integrin in the luminal epithelium. CONCLUSIONS: As these integrins have been shown to be important in implantation their reduced expression after IVF treatment may have an adverse effect on pregnancy rates.     

Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma

Eosinophilic inflammation is a characteristic feature of asthma. Integrins are highly versatile cellular receptors that regulate extravasation of eosinophils from the postcapillary segment of the bronchial circulation to the airway wall and airspace. Such movement into the asthmatic lung is described as a sequential, multistep paradigm, whereby integrins on circulating eosinophils become activated, eosinophils tether in flow and roll on bronchial endothelial cells, integrins on rolling eosinophils become further activated as a result of exposure to cytokines, eosinophils arrest firmly to adhesive ligands on activated endothelium, and eosinophils transmigrate to the airway in response to chemoattractants. Eosinophils express seven integrin heterodimeric adhesion molecules: alpha 4 beta 1 (CD49d/29), alpha 6 beta 1 (CD49f/29), alpha M beta 2 (CD11b/18), alpha L beta 2 (CD11a/18), alpha X beta 2 (CD11c/18), alpha D beta2 (CD11d/18), and alpha 4 beta 7 (CD49d/beta 7). The role of these integrins in eosinophil recruitment has been elucidated by major advances in the understanding of integrin structure, integrin function, and modulators of integrins. Such findings have been facilitated by cellular experiments of eosinophils in vitro, studies of allergic asthma in humans and animal models in vivo, and crystal structures of integrins. Here, we elaborate on how integrins cooperate to mediate eosinophil movement to the asthmatic airway. Antagonists that target integrins represent potentially promising therapies in the treatment of asthma.     

Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of alpha6 integrin and low levels of CD71 (alpha6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human alpha6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not.     

Integrin beta4, keratinocytes and papillomavirus infection

Integrin beta4 is a transmembrane protein expressed predominantly on epithelial cells. In human epidermis integrin beta4 associates with integrin beta6. Integrin alpha6beta4 is concentrated at the basement membrane zone, where it localizes to specialized adhesion structures called hemidesmosomes. In addition to its adhesive functions, keratinocyte integrin beta4 has been identified as an important regulator of epidermal homeostasis. This review summarizes the current knowledge regarding the role of integrin beta4 in keratinocyte adhesion, migration, as well as growth and differentiation. Changes in integrin beta4 expression in pathological conditions in skin and mucosa, especially those associated with human papillomavirus infection, are described.     

Loss of co-localization of alpha 6 beta 4 integrin and collagen VII in bladder cancer.

In normal epithelial cells, the alpha 6 beta 4 integrin co-localizes with the hemidesmosomal anchoring complex on the basolateral surface of basal cells. We studied the co-expression of alpha 6 beta 4 integrin with collagen VII, a component of the hemidesmosomal anchoring complex, in normal bladder tissues and in bladder cancers. In normal bladder, the alpha 6 beta 4 integrin co-localized with collagen VII at the junction of the basolateral surface of the basal urothelial cells and the lamina propria. In five of six noninvasive bladder cancers, the localization of collagen VII remained unchanged, found at the junction of the basal cells and the papillary connective tissue. However, in these tumors the alpha 6 beta 4 expression was not polarized and was expressed on suprabasal as well as basal cells. In invasive bladder cancers, the majority (25 of 30) showed either loss of alpha 6 beta 4 and/or collagen VII expression or showed a lack of co-localization of alpha 6 beta 4 and collagen VII. Our results show derangement of the co-localization of these two components of the hemidesmosomal anchoring complex is a consistent event in bladder cancer. Furthermore, the degree of derangement increases in invasive cancers. Loss of co-expression and co-localization of alpha 6 beta 4 and collagen VII may predispose cancer cells to local invasion and may facilitate metastasis as well.     

Expression of epithelial adhesion proteins and integrins in chronic inflammation.

Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma.     

Clomiphene citrate does not affect the secretion of alpha3, alphaV and beta1 integrin molecules during the implantation window in patients with unexplained infertility.

BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of beta1, alpha3 and alphaV integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5-9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of alpha(v) but not beta1 and alpha3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 +/- 0.12 versus 2.21 +/- 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of alpha(v), beta1 and alpha3 integrin molecules, which might play a role in implantation, was not affected.     

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.