Glucose-Induced Alteration of Integrin Expression and Function in Cultured Human Mesangial Cells

Alteration in mesangial volume, due to an increase of the matrix surrounding mesangial cells, is a hallmark indicator of nephropathy in diabetes. Mesangial cells may also play a significant role in the development of nephropathy. Therefore, we examined the effect of glucose on the expression of integrins by cultured human mesangial cells and their ability to interact with collagen IV, a major component of the mesangial matrix. Human mesangial cells were grown in 5 and 25 mM glucose and their integrin profile was examined by immunoprecipitation and flow cytometry in each experimental condition. The results indicate that when mesangial cells were grown in 25 mM glucose, the expression of integrin subunit α2, was increased, while the α1 subunit was considerably decreased, as compared to cells grown in 5 mM glucose. Additionally, mesangial cells were tested for their ability to adhere to collagen IV in a solid-phase assay in the presence of neutralizing antibodies to integrin subunits. The results of these experiments indicate that both α1 and α2 complexed to β1 (α2β1 and α1β1) are major mesangial cell receptors for adhesion to collagen IV both in 5 and 25 mM glucose. The two receptors act in concert to mediate adhesion of mesangial cells to type IV collagen. When cell surface expression of the α1 subunit in 25 mM glucose was reduced, the α2 subunit was involved in adhesion to a greater extent than it was in 5 mM glucose. Immunoperoxidase histochemical studies localized both α1 and α2 integrin subunits in the mesangium of normal adult kidneys, suggesting that in vivo interaction with collagen IV could involve both of these receptors. These observations suggest that glucose-induced alterations in integrin expression may modify the ability of mesangial cells to interact with collagen IV.     

Effects of glycated albumin on mesangial cells: evidence for a role in diabetic nephropathy

Nonenzymatically glycated proteins are preferentially transported across the glomerular filtration barrier, and the glomerular mesangium in diabetes is bathed with serum containing increased concentrations of glycated albumin. We investigated effects of glycated albumin on mesangial cells, which are involved in diabetic nephropathy. [³H]-thymidine incorporation was significantly inhibited when murine mesangial cells were grown in culture media containing human serum that had been nonenzymatically glycated by incubation for 4 days with 28 mM glucose. This inhibition was reversed when monoclonal antibodies that selectively react with Amadori products of glycated albumin were added to the culture media. Purified glycated albumin containing Amadori adducts of the glycation reaction induced significant inhibition of thymidine incorporation and stimulation of Type IV collagen secretion compared with cells cultured in the presence of purified nonglycated albumin. These changes were prevented when monoclonal antibodies specifically reactive with fructosyl-lysine epitopes in glycated albumin were added to the cultures. The antibodies had no effect on growth or collagen production in the presence of nonglycated albumin. The results provide the first evidence directly implicating Amadori adducts in glycated albumin in the pathogenesis of diabetic nephropathy, which is characterized by decreased cellularity in association with expansion of the mesangial matrix.     

Intermittent high glucose enhances cell proliferation and VEGF expression in retinal endothelial cells: the role of mitochondrial reactive oxygen species

Proliferation of human retinal endothelial cells (HRECs) is an important event in the development of diabetic retinopathy. Glucose fluctuations are strong predictor of diabetic vascular complications. In this study we have investigated the effect of intermittent high glucose on proliferation and expression of vascular endothelial growth factor (VEGF) in HRECs. The possible involvement of mitochondrial reactive oxygen species (ROS) was assessed. HRECs were incubated for 72 h in media containing different glucose concentrations: 5, 25, 5 mmol/l alternating with 25 mmol/l glucose, with or without Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) and thenoyltri-fluoroacetone (TTFA). The cell proliferation, VEGF expression, mitochondrial ROS, nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) were measured. In cultured HRECs, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time-dependent manner. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, mRNA and protein levels of VEGF compared with HRECs treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose. The levels of mitochondrial ROS, nitrotyrosine and 8-OhdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA can effectively prevent cell proliferation and overexpression of VEGF, as well as overproduction of mitochondrial ROS, nitrotyrosine and 8-OhdG in HRECs induced by constant or intermittent high glucose. Intermittent high glucose enhances cell proliferation and overexpression of VEGF through reactive oxygen species (ROS) overproduction at the mitochondrial transport chain level in HRECs, indicating that glycemic variability have important pathological effects on the development of diabetic retinopathy dependent of mitochondrial ROS.     

Albumin modified by Amadori glucose adducts activates mesangial cell type IV collagen gene transcription

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited³H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.     

Glycated albumin modified by amadori adducts modulates aortic endothelial cell biology

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited³H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC₅₀ value of 46 ± 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [³H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation,but is independent of post-translational modifications of osteopontin

Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn^2 +: the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C-terminus of the α4 integrin-binding site also did not affect binding affinity. THP-1 cells express a low-affinity conformation of the integrin and adhered to OPN only in the presence of Mn^2 + plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands without integrin activation. Studies with ligand-induced binding site antibodies demonstrated that the SVVYGLR peptide of OPN bound to the α4 integrin with a similar affinity as the LDV peptide of fibronectin, suggesting that a high off-rate is responsible for the reduced binding of OPN to the low-affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions.     

Osteopontin is a mediator of the lateral migration of neuroblasts from the subventricular zone after focal cerebral ischemia

We and others have shown that focal cerebral ischemia induces lateral migration of neuroblasts from the ipsilateral subventricular zone (SVZ) to the ischemic striatum. The signaling pathways underlying this phenomenon are not fully understood. The present study examined the role of osteopontin (OPN) in post-ischemic lateral migration of neuroblasts. Focal ischemia was induced by transient middle cerebral artery occlusion in adult spontaneous hypertensive rats. The expression of OPN in the ischemic brain was evaluated by immunohistochemistry, which showed that an up-regulation of OPN expression in the ipsilateral striatum at day 3, 7, 14 and 1 month of reperfusion with a peak at day 7. Double staining showed co-localization of OPN with ED1⁺ macrophages/microglia in the ischemic regions. Inhibition of OPN activity by infusing a neutralizing antibody against OPN into the ischemic striatum significantly decreased the area covered with doublecortin⁺ neuroblasts in the ipsilateral striatum. In vitro, OPN treatment did not affect the proliferation of neural progenitors, but induced an increased trans-well and radial migration of neural progenitors. The cultured neural progenitors expressed the OPN receptors CD44 and integrin β₁. Blockade of the CD44 receptor had no effects on OPN mediated trans-well and radial migration of neural progenitors. However, blockade of integrin β₁ receptor abolished the migration of neural progenitors in the absence or the presence of OPN. These results suggest that up-regulated expression of OPN produced by macrophages/microglia in the ischemic brain is an attractant and inducer for the lateral migration of neuroblasts from the SVZ to the injured region.     

Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition

Diabetic nephropathy, one of the microvascular complications of diabetes mellitus, is a leading cause of end-stage renal disease. Berberine is one of the main constituents of Coptidis Rhizoma and Cortex Phellodendri. In this study, we investigated the effects of berberine on fibronectin and collagen production, and explored the role of p38MAPK signaling pathway in rat glomerular mesangial cells cultured under high glucose condition. Six groups were divided according to the different experimental conditions: (1) Normal glucose group (NG); (2) Mannitol group (Mannitol); (3) High glucose group (HG); (4) SB203580 treatment group (HG + SB203580); (5) Berberine low dosage group (HG + BBR 30 μM); (6) Berberine high dosage group (HG + BBR 90 μM). Cell proliferation and collagen synthesis were measured by MTT and ³H-proline incorporation assay, respectively. The phospho-p38MAPK, phospho-cAMP response element binding protein (CREB) and fibronectin were detected by western blot analysis. Fibronectin protein expression and collagen synthesis were significantly increased in HG-treated group compared with normal glucose group (P < 0.05). In SB203580 treatment group and two groups of berberine, protein expression of fibronectin and collagen synthesis were obviously decreased compared with HG-treated group (P < 0.05). Berberine significantly decreased protein expression of fibronectin compared with SB203580 treatment group (P < 0.05). Berberine at high dosage significantly decreased collagen synthesis compared with SB203580 treatment group (P < 0.05). Both SB203580 and berberine significantly decreased phospho-p38MAPK and phospho-CREB level compared with HG-treated group (P < 0.05). These results indicated that berberine might inhibit fibronectin and collagen synthesis partly via p38MAPK signal pathway in rat glomerular mesangial cells exposed to high glucose.     

Phytolacca americana inhibits the high glucose-induced mesangial proliferation via suppressing extracellular matrix accumulation and TGF-beta production.

This study describes a potential of Phytolaccaceae (Phytolacca americana var.) as an inhibitor of high glucose-stimulated production of extracellular matrix (ECM) proteins and TGF-beta in cultured glomerular mesangial cells (GMCs). Raising the ambient glucose concentration for 24 hrs caused a dose-dependent increase in [3H]thymidine incorporation of GMCs, and the maximal response was achieved at 20 mM. Phytolaccaceae extracts (2.5-20 microg/ml) inhibited the high glucose-induced [3H]thymidine incorporation in a dose-dependent manner, and the concentrations tested here did not affect to the cell viability. Exposure of the GMCs to 20 mM glucose caused both ECM (collagen and fibronectin) accumulation and TGF-beta secretion, and these changes were significantly diminished by treatment of GMCs with Phytolaccaceae (10 microg/ml). Taken together, these results indicate that Phytolaccaceae inhibits the high glucose-induced GMCs proliferation partially through suppressing accumulation of ECM components and TGF-beta production, suggesting that Phytolaccaceae may be a promising agent for treating the development and progression of diabetic glomerulopathy.     

Control of cell proliferation via elevated NEDD8 conjugation in oral squamous cell carcinoma

Adenosine (ADO) is an intermediary metabolite of adenosine trisphosphate degradation and a vasoactive mediator. We showed previously that ADO induces contraction and proliferation in rat mesangial cells by a mechanism involving A1 and A2 receptors. The studies concerning the effect of ADO on extracellular matrix (ECM) accumulation in mesangial cells are scarce. The purpose of our study was to evaluate the effect of ADO and the effect of the selective stimulation of A1 and A2 ADO receptors on the expression of ECM components fibronectin and collagen type I, in human and rat renal mesangial cells. Cultured human and rat renal mesangial cells were subjected to selective stimulation of A1 and A2 ADO receptors for 24 and 48 h. Fibronectin and collagen type I expression was evaluated by Western blot; total collagen synthesis was measured by [³H]-proline incorporation into collagen proteins. ADO, A1 and A2 receptor stimulation induce increases in fibronectin expression in rat mesangial cells, and A1 receptor stimulation partially inhibits fibronectin expression in serum-stimulated rat mesangial cells, without any effect in human mesangial cells. A2 receptor stimulation reduces collagen type I expression in serum-stimulated mesangial cells. Neither ADO nor A1 or A2 receptor stimulation induce significant changes in total collagen synthesis. These data suggest that ADO is not a major regulator of ECM synthesis in rat and human mesangial cells.     

Glucose deficiency reduces collagen synthesis in breast cancer MCF7 cells

We decided to study the effect of glucose deprivation on collagen metabolism in MCF7 cells. The incorporation of [³H]‐proline into collagenase‐sensitive and hydroxyproline‐containing proteins was used as an index of collagen synthesis, whereas pulse—chase technique was employed to evaluate the degradation of newly synthesized proteins. The MCF7 cells incubated in high glucose medium synthesized detectable amounts of collagenous proteins. Most of them were found in the cell layer. The shortage of glucose resulted in about 30% reduction in collagen synthesis. The pulse—chase experiments demonstrated that proportionally less collagen was degraded in cultures incubated in low‐glucose than in high‐glucose media.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either βl, α3, α5, α6 or β3 integrin subunits. Increased phosphorylation of the 100–130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100–130 kDa complex was identified as the focal adhesion tyrosine kinase p125^FAK. The phosphorylation of the pl25^FAK was also observed by inducing βl integrin clustering in rum adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Rat mesangial cell-matrix interactions in culture

The glomerular mesangium contains fibronectin (FN), laminin, and collagen IV, but it remains unclear whether these matrix proteins affect mesangial cellular functions. The present experiments were designed to test whether cell-matrix interactions could affect some functions of mesangial cells. Cultured rat mesangial cells synthesized a cellular form of FN that was both secreted and incorporated into an extensive, fibrillar pericellular matrix. This FN matrix was increased in high-density cultures and was more developed in human mesangial cells. Rat mesangial cells in vitro displayed a marked capacity to incorporate exogenous FN into a pericellular matrix, demonstrating that accumulations of FN in the mesangial matrix could result from endogenous and/or exogenous sources. Rat mesangial cells also expressed RGD-sensitive integrin receptors for FN, laminin, and collagens I and IV that promoted cell adhesion and that directed differential changes in morphology. Indirect evidence suggested the existence of other mesangial binding sites for extracellular matrix proteins. FN and collagen IV also stimulated modest increases in [3H]thymidine uptake and cell number by quiescent cells. Taken together, these results suggest that cultured mesangial cells present a model system for studying the regulation of cell-matrix interactions in the mesangium.     

Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

Osteopontin is a mediator of the lateral migration of neuroblasts from the subventricular zone after focal cerebral ischemia

We and others have shown that focal cerebral ischemia induces lateral migration of neuroblasts from the ipsilateral subventricular zone (SVZ) to the ischemic striatum. The signaling pathways underlying this phenomenon are not fully understood. The present study examined the role of osteopontin (OPN) in post-ischemic lateral migration of neuroblasts. Focal ischemia was induced by transient middle cerebral artery occlusion in adult spontaneous hypertensive rats. The expression of OPN in the ischemic brain was evaluated by immunohistochemistry, which showed that an up-regulation of OPN expression in the ipsilateral striatum at day 3, 7, 14 and 1 month of reperfusion with a peak at day 7. Double staining showed co-localization of OPN with ED1⁺ macrophages/microglia in the ischemic regions. Inhibition of OPN activity by infusing a neutralizing antibody against OPN into the ischemic striatum significantly decreased the area covered with doublecortin⁺ neuroblasts in the ipsilateral striatum. In vitro, OPN treatment did not affect the proliferation of neural progenitors, but induced an increased trans-well and radial migration of neural progenitors. The cultured neural progenitors expressed the OPN receptors CD44 and integrin β₁. Blockade of the CD44 receptor had no effects on OPN mediated trans-well and radial migration of neural progenitors. However, blockade of integrin β₁ receptor abolished the migration of neural progenitors in the absence or the presence of OPN. These results suggest that up-regulated expression of OPN produced by macrophages/microglia in the ischemic brain is an attractant and inducer for the lateral migration of neuroblasts from the SVZ to the injured region.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.