IFATS collection: Adipose-derived stromal cells improve the foreign body response

Many implanted devices fail due to the formation of an avascular capsule surrounding the device. Additionally, fat has long been known to promote healing and vascularization. The goals of this study were to identify potential mechanisms of the provascular actions of adipose-derived stromal cells (ASCs) and to improve implant biocompatibility. First, adult ASCs and fibroblasts from rats were attached to polyurethane and polystyrene in vitro and their cytokine secretion profile was analyzed. Secretion of vascular endothelial growth factor (VEGF) from ASCs was 10-70 times higher than fibroblasts after 3 and 6 days. Next, polyurethane, bare and with cellular coatings, was implanted subcutaneously in rats. The fibrous capsule surrounding bare polyurethane implants was 17%-32% thicker and the amount of collagen was 27% greater than the capsule surrounding ASC-coated implants. Finally, the microvessel density adjacent to ASC-coated polyurethane was approximately 50%-80% higher than bare polyurethane. In summary, ASCs attached to polyurethane have a dramatically increased VEGF production compared with fibroblasts in vitro, and these cells also produce an increased microvessel density in the surrounding tissue when implanted subcutaneously in rats. Disclosure of potential conflicts of interest is found at the end of this article.     

IFATS collection: Adipose stromal cells adopt a proangiogenic phenotype under the influence of hypoxia

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.     

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.     

IFATS collection: Using human adipose-derived stem/stromal cells for the production of new skin substitutes

The ability to harvest and culture stem cell populations from various human postnatal tissues is central to regenerative medicine applications, including tissue engineering. The discovery of multipotent mesenchymal stem cells within the stromal fraction of adipose tissue prompted their use for the healing and reconstruction of many tissues. Here, we examined the influence of adipose-derived stem/stromal cells (ASCs) on skin's regenerative processes, from a tissue engineering perspective. Using a self-assembly approach, human skin substitutes were produced. They featured a stromal compartment containing human extracellular matrix endogenously produced from either dermal fibroblasts or adipose-derived stem/stromal cells differentiated or not toward the adipogenic lineage. Human keratinocytes were seeded on each stroma and cultured at the air-liquid interface to reconstruct a bilayered skin substitute. These new skin substitutes, containing an epidermis and a distinctive stroma devoid of synthetic biomaterial, displayed characteristics similar to human skin. The influence of the type of stromal compartment on epidermal morphogenesis was assessed by the evaluation of tissue histology, the expression of key protein markers of the epidermal differentiation program (keratin [K] 14, K10, transglutaminase), the expression of dermo-epidermal junction components (laminins, collagen VII), and the presence of basement membrane and hemidesmosomes. Our findings suggest that adipose-derived stem/stromal cells could usefully substitute dermal fibroblasts for skin reconstruction using the self-assembly method. Finally, by exploiting the adipogenic potential of ASCs, we also produced a more complete trilayered skin substitute consisting of the epidermis, the dermis, and the adipocyte-containing hypodermis, the skin's deepest layer. Disclosure of potential conflicts of interest is found at the end of this article.     

An arthritogenic alphavirus uses the alpha1beta1 integrin collagen receptor

Ross River (RR) virus is an alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis or RR virus disease. Here we provide evidence that RR virus uses the collagen-binding alpha1beta1 integrin as a cellular receptor. Infection could be inhibited by collagen IV and antibodies specific for the beta1 and alpha1 integrin proteins, and fibroblasts from alpha1-integrin-/- mice were less efficiently infected than wild-type fibroblasts. Soluble alpha1beta1 integrin bound immobilized RR virus, and peptides representing the alpha1beta1 integrin binding-site on collagen IV inhibited virus binding to cells. We speculate that two highly conserved regions within the cell-receptor binding domain of E2 mimic collagen and provide access to cellular collagen-binding receptors.     

The alpha2beta1 integrin: a novel collectin/C1q receptor

Our laboratory focuses on the alpha2beta1 integrin, a receptor for a number of matrix and non-matrix ligands, including collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1 (MMP-1), endorepellin, and several viruses. The alpha2beta1 integrin is expressed on numerous different cell types, including epithelial cells, endothelial cells, fibroblasts, and hematopoietic elements, including platelets and specific subsets of leukocytes. Although alpha2beta1 integrin expression is widespread, it is not ubiquitous. Rather, it is expressed in a differentiation-dependent and activation-dependent manner. Interactions between the alpha2beta1 integrin and extracellular matrix ligands have been implicated in important biological processes including inflammation and immunity. Studies from a number of laboratories have demonstrated a role for the alpha2beta1 integrin during the immune response. Our laboratory generated an alpha2beta1 integrin-deficient mouse to define the role of the alpha2beta1 integrin in vivo. Our studies demonstrated that the alpha2-null mice have a profound defect in the innate immune response. We have recently reported the identification of a novel family of ligands for the alpha2beta1 integrin, which include C1q and the collectins. The goal of this article is to review the important role that the interaction between the alpha2beta1 integrin and C1q plays in the innate immune response. The identification of C1q and the collectins as ligands for the alpha2beta1 integrin suggests that the integrin may play important roles in a number of immunological responses.     

Cytomegalovirus-infected neuroblastoma cells exhibit augmented invasiveness mediated by beta1alpha5 integrin (VLA-5)

Previously, experimental in vivo results showed that the productively and persistently human cytomegalovirus (HCMV)-infected neuroblastoma cell line UKF-NB-4AD169 exhibits a more malignant phenotype than the non-infected variant UKF-NB-4. To prove the assumption that enhanced malignancy may be due to enhanced invasive potential of the infected cells we studied interactions of both lines with monolayers of cultured endothelial cells. UKF-NB-4AD169 cells adhered to and transmigrated through endothelial monolayer to a significantly higher extent compared with UKF-NB4. Furthermore, the adhesion of UKF-NB-4AD169 but not of UKF-NB4 resulted in focal disruption of the monolayer integrity which facilitates tumor cell transmigration. Blocking antibodies directed against the beta1 integrin chain as well as beta1alpha5 on the tumor cells specifically inhibited adhesion in a concentration-dependent manner. When UKF-NB-4 were pretreated with a beta1 integrin activating antibody, focal disruption of the endothelial integrity also occurred. These findings lead us to suggest that HCMV infection activates beta1alpha5 in the host neuroblastoma cell which in turn enables these cells to tightly adhere to endothelial cells. In the presence of the protease inhibitor phenantroline, beta1alpha5-mediated adhesion was not impaired whereas UKF-NB4AD169-mediated endothelial monolayer permeabilization was dose dependently inhibited. We conclude that human cytomegalovirus infection contributes to augmented neuroblastoma invasiveness via adhesion of activated beta1alpha5 and subsequent matrix digestion by proteases.     

HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120, a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells, engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.     

IFATS collection: The role of human adipose-derived stromal cells in inflammatory microvascular remodeling and evidence of a perivascular phenotype

A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of platelet-derived growth factor B chain (PDGF-BB) and vascular endothelial growth factor 165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. Ten, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared with 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle alpha-actin (10%) and neuron-glia antigen 2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels. Disclosure of potential conflicts of interest is found at the end of this article.     

Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor.

The alpha 2 beta 1 integrin (platelet membrane glycoprotein Ia-IIa, VLA-2, ECMR-II) functions as a cell surface receptor for collagen. The authors have determined the histologic distribution of the alpha 2 beta 1 receptor in normal tissues by immunohistochemical technique. The studies revealed that the alpha 2 beta 1 receptor was expressed on fibroblasts, endothelial cells, and epithelial cells from multiple sites including skin, tonsil, breast, sweat gland, gastrointestinal tract, lung, bladder, cervix, and prostate. Follicular dendritic cells of the lymph node, tonsil, and spleen and dendritic cells of the thymus also expressed the alpha 2 beta 1 receptor. The receptor also was present on Schwann cells of ganglia and on neuroglia. Greatly enhanced expression of the receptor in regions of proliferating epithelium suggests that enhanced expression of alpha 2 beta 1 is associated with orderly, regulated cell proliferation. The circumferential staining pattern of the alpha 2 beta 1 integrin within many epithelia is virtually identical to that observed for other adhesive receptors, such as the cadherins, which have been implicated in cell-cell adhesion.     

Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.

We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.     

The alpha v beta 3 integrin as a tumor homing ligand for lymphocytes

Despite the presence of tumor-specific effector cells in the circulation of cancer patients, the immune response of the majority of these patients is not sufficient to prevent the growth and spread of their tumors. That tumor cells can be killed in vitro by tumor-reactive cytotoxic T cells is testimony to the fact that the tumors are not inherently resistant to T cell killing, but rather that there is a failure in immune recognition and effector cell activation. Many reasons for this failure of the body's defense system have been suggested, including the inability of tumor-reactive lymphocytes to migrate to tumor tissue. Here we designed a strategy to improve homing of primary lymphocytes into vascularized tumors. As a homing molecule we selected the integrin alpha v beta 3 since it is expressed by angiogenic vascular endothelium in tumors. To promote lymphocyte adhesion to alpha v beta 3 we "painted" primary lymphocytes with a recombinant, glycosylphosphatidylinositol-linked high-affinity ligand for alpha v beta 3. These painted lymphocytes specifically bound to alpha v beta 3 in vitro and homed to vascularized, solid tumors in vivo. This novel strategy may provide a significant advance in anti-tumor treatment such as adoptive immune therapy.     

Invasion of human respiratory epithelial cells by Bordetella pertussis: possible role for a filamentous hemagglutinin Arg-Gly-Asp sequence and alpha5beta1 integrin

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human alpha5beta1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell alpha5beta1 integrin.     

Basement membrane laminin-5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for alpha3beta1 integrin

Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors alpha2beta1, alpha3beta1 and alpha6beta4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immunoreactivity for chains of Ln-5 (alpha3-beta3-gamma2) and Ln-10 (alpha5-beta1-gamma1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln al chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for alpha2, alpha6, beta1 and beta4 Ints was found in all tumors and that for alpha3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the gamma2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to alpha3 and beta1 Int subunits, but not those to Int alpha2 or alpha6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln-5, produced by carcinoma cells, is a major BM component and the cells use the alpha3beta1 Int complex for adhesion to Ln-5.     

整合素α5β1在成骨细胞与生物衍生材料黏附过程中的表达

背景：整合素在细胞与材料的黏附过程中发挥重要作用。 目的：了解整合素α5β1在成骨细胞构建组织工程骨和组织工程骨膜过程中的表达,探讨在成骨细胞与生物衍生材料黏附过程中整合素α5β1发挥的作用。 方法：选用人胚骨膜来源成骨细胞为种子细胞,接种生物衍生骨及羊膜支架材料上,制备组织工程骨及骨膜,分别培养2,4,6,8,10 d。单纯培养的成骨细胞作为对照组。 结果与结论：实时定量PCR测定结果,培养早期整合素α5表达呈阴性,整合素β1组织工程骨中的表达略高于组织工程骨膜,培养2,6 d组比较差异有显著性意义。单纯细胞培养的对照组中,整合素β1稳定,各时间段比较差异无显著性意义。提示生物衍生材料有利于成骨细胞黏附,但在黏附过程的初期,成骨细胞可能是通过纤维连接蛋白以外的其他蛋白质完成该过程的。     

Dynamic expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors by human vascular smooth muscle cells. Alpha 2 beta 1 integrin is required for chemotaxis across type I collagen-coated membranes.

Vascular smooth muscle cells (SMCs) in the media of normal arteries express alpha 1 beta 1 integrin with no detectable alpha 2 beta 1 as determined by immunocytochemistry. In contrast, immunoprecipitation of integrins expressed by human SMCs cultured from medial explants shows strong expression of alpha 2 beta 1 and no expression of alpha 1 beta 1. The apparent reciprocal expression of these two collagen and laminin receptors was confirmed by flow cytometric analysis of fluorescent labeled cells. Freshly isolated SMCs had detectable alpha 1, alpha 3, alpha 5, and alpha v subunits with low levels of detectable beta 3 and no detectable alpha 2. Cultured SMCs expressed alpha 2, alpha 3, alpha 5 and alpha v subunits with little or no alpha 1 or beta 3. Neither alpha 4 nor alpha 6 were detectable in freshly isolated or cultured cells. Expression of alpha 2 beta 1 receptors by cultured SMCs appears to be required for the migration of these cells on type I collagen. Migration of cultured SMCs across a type I collagen-coated membrane toward two different chemotactic stimuli, platelet-derived growth factor-BB (1 nmol/L) and insulin-like growth factor-(1 nmol/L), was Mg2+ dependent and inhibited by preincubation of cells with an affinity-purified polyclonal anti-alpha 2 beta 1 antibody or by monoclonal antibodies directed against the individual alpha 2 or beta 1 subunits. Attachment to type 1 collagen membranes was not affected by antibodies under conditions where migration was significantly impeded. The combined data show that SMC expression of alpha 1 beta 1 and alpha 2 beta 1 integrin receptors for collagen and laminin is dynamic and reciprocal and may be important with respect to SMC migration on type I collagen. These findings are potentially important in understanding the pathophysiology of vascular diseases, for example, atherosclerosis and restenosis following balloon angioplasty, where SMC migration is a contributing factor.     

Uniform overexpression and rapid accessibility of alpha5beta1 integrin on blood vessels in tumors

Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.     

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2). The expression of alpha2beta1 conferred YBmalpha2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmalpha2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmalpha2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for alpha4beta1 integrin. The interaction of alpha4beta1 with VCAM-1 was further stimulated upon ligation with alpha2beta1-specific mAb. The use of specific inhibitory mAb demonstrated the role of alpha4beta1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of alpha2beta1 differentially regulated alpha4alpha1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of alpha2beta1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of alpha2beta1 enhanced alpha4beta1-mediated extravasation of YBmalpha2 cells in the liver.