Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen

Mouse endochondral chondrocytes were immortalized with a temperature- sensitive simian virus 40 large tumor antigen. Several clonal isolates as well as pools of immortalized cells were characterized. In monolayer cultures at the temperature permissive for the activity of the large tumor antigen (32 degrees C), the cells grew continuously with a doubling time of approximately 2 d, whereas they stopped growing at nonpermissive temperatures (37 degrees C-39 degrees C). The cells from all pools and from most clones expressed the genes for several markers of hypertrophic chondrocytes, such as type X collagen, matrix Gla protein, and osteopontin, but had lost expression of type II collagen mRNA and failed to be stained by alcian blue which detects cartilage- specific proteoglycans. The cells also contained mRNAs for type I collagen and bone Gla protein, consistent with acquisition of osteoblastic-like properties. Higher levels of mRNAs for type X collagen, bone Gla protein, and osteopontin were found at nonpermissive temperatures, suggesting that the expression of these genes was upregulated upon growth arrest, as is the case in vivo during chondrocyte hypertrophy. Cells also retained their ability to respond to retinoic acid, as indicated by retinoic acid dose-dependent and time- dependent increases in type X collagen mRNA levels. These cell lines, the first to express characteristic features of hypertrophic chondrocytes, should be very useful to study the regulation of the type X collagen gene and other genes activated during the last stages of chondrocyte differentiation.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells

 Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G₀ phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G_1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G_1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs. Accepted: 22 November 1996     

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.     

Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast-like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum-starved stromal cells, PDGF stimulated [³H]-thymidine incorporation into DNA in a dose-dependent manner with a maximum stimulation of 15-fold at 500 ng/ml. By comparison, insulin-like growth factor (IGF-I) has a small effect on [³H] -thymidine incorporation. The effect of PDGF and IGF-I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1-, 2.3-, and 14-fold, respectively. Addition of PDGF to dexamethasone-treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)₂D₃. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF-I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF-I. Furthermore, PDGF attenuated the stimulatory effect of IGF-I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF-I increased [³H]-thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent. © 1995 Wiley-Liss, Inc.     

miR-485 suppresses inflammation and proliferation of mesangial cells in an in vitro model of diabetic nephropathy by targeting NOX5

Diabetic nephropathy (DN) is among the common complications of diabetes and is a major cause of end-stage kidney disease. Emerging data indicate that renal inflammation is involved in DN progression and aggravation. Still, the exact cellular mechanisms remain unclear. Dysregulated expression of microRNAs (miRNAs) is associated with multiple diseases, including DN. The relationship between miRNAs and inflammation in DN is also unexplored. Here, we evaluated the role of miR-485 in mediating the response of human mesangial cells (HMCs) to a high glucose (HG) concentration, and the potential underlying mechanism. We found that miR-485 expression is significantly decreased in HG-stimulated HMCs. Overexpression of miR-485 suppressed HG-induced proliferation of HMCs. Lower production of proinflammatory cytokines (i.e., TNF-α, IL-1β, and IL-6) was observed in miR-485–overexpressing HMCs. Overexpression of miR-485 markedly suppressed the overexpression of extracellular-matrix proteins, e.g., collagen IV (Col IV) and fibronectin (FN), in HG-stimulated HMCs. Furthermore, miR-485 suppressed the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5), restrained the HG-induced HMC proliferation, downregulated the expression of proinflammatory cytokines, and inhibited the production of extracellular-matrix proteins in HMCs. These results provide new insights into the involvement of the miR-485–NOX5 signaling pathway in DN progression.     

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.     

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, cell number, [³H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of vasopressin on type IV collagen production in human mesangial cells

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V_1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-β secretion by human mesangial cells and this effect was inhibited by V_1A receptor antagonists. Furthermore, TGF-β also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-β antibody. These findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-β synthesis mediated by V_1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.     

Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells

Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [³H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [³H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.     

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.     

9HODE stimulates cell proliferation and extracellular matrix synthesis in human mesangial cells via PPARgamma

Plasma oxidized low-density lipoprotein (OX-LDL) levels are elevated in patients with renal diseases, including diabetic nephropathy. We examined effects of OX-LDL on cell proliferation and extracellular matrix (ECM) production by using normal human mesangial cells. Furthermore, we examined possible involvement of peroxisome proliferator-activated receptor gamma (PPARgamma). Mesangial cell proliferation with OX-LDL, 9-hydroxy-10,12-octadecadienoic acid (9HODE), and 13-hydroxy-9,11-octadecadienoic acid (13HODE), the major components of OX-LDL, were determined by 5-bromo-2'-deoxyuridine (BrdU) or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) incorporation. The effect of OX-LDL on mesangial cell proliferation with PD98059 pretreatment was determined by BrdU incorporation. Type IV collagen, fibronectin, and PPARgamma expression with OX-LDL or 9HODE or 13HODE was determined by Western blotting. Type IV collagen expression with antisense oligonucleotide against PPARgamma pretreatment was also determined by Western blotting. The effect of PD98059 pretreatment on PPARgamma expression was determined by Western blotting. In mesangial cells exposed to isolated OX-LDL from human plasma, BrdU incorporation was increased, and this increase was deleted by PD98059. Type IV collagen expression was significantly increased by OX-LDL. 9HODE and 13HODE increased BrdU and MTT incorporation into mesangial cells and also increased expressions of Type IV collagen and fibronection, the major components of ECM. PPARgamma expression in mesangial cells was stimulated by 9HODE. The reduction of PPARgamma synthesis by pretreatment of antisense oligonucleotide against PPARgamma remarkably attenuated Type IV collagen synthesis induced by 9HODE. PPARgamma expression induced by 9HODE was also reduced by PD98059 pretreatment. These findings demonstrate that 9HODE, the major component of OX-LDL, stimulates cell proliferation and ECM production of human mesangial cells. In addition, the stimulatory effects are, at least in part, mediated by PPARgamma, which may exist in downstream of ERK1/2 pathway.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Desmin expression in fibroblasts of murine periovular granuloma during liver Schistosoma mansoni infection

We have studied the expression of the desmin gene, a muscle-specific intermediate filament protein in the granuloma cells of mouse liver infected with Schistosoma mansoni. In situ hybridization using a desmin DNA probe showed that fibroblastic cells in the granuloma strongly expressed desmin mRNAs, while in normal liver these cells did not express this mRNA to a detectable degree. The quantitative analysis of total RNAs demonstrated that the proportion of specific desmin mRNA increased from 14 to 18 weeks after infection and decreased at 20 weeks. The analysis of collagen gene expression indicated that the amount of type III collagen mRNAs was still increasing after 18 weeks from infection; in contrast, the amount of type I collagen mRNAs remained unchanged at that stage. A good correlation was observed between the detection of the specific mRNAs and the detection of both desmin and collagen molecules. Therefore, these data point to a coordinate induction of desmin and collagen gene expression during Schistosomal granuloma formation. They also suggest that the expression of the myofibroblast phenotype involves the induction of both genes.     

A vitamin D analog regulates mesangial cell smooth muscle phenotypes in a transforming growth factor-beta type II receptor-mediated manner.

Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-beta (TGF-beta) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D(3) (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) in a dose-dependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle alpha actin mRNA, but 1, 25(OH)(2)D(3)-treated MC-E cells did not. The addition of a TGF-beta(1)-neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels. However, after exposure to 1, 25(OH)(2)D(3) or OCT, there was no significant difference in the secretion of active TGF-beta. We next investigated whether TGF-beta type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.     

Human-derived vascular smooth muscle cells produce angiotensin II by changing to the synthetic phenotype

We investigated whether vascular smooth muscle cells (VSMC)-derived from human produce angiotensin (Ang) II upon change from the contractile phenotype to the synthetic phenotype by incubation with fibronectin (FN). Expression of alpha-smooth muscle (SM) actin, apparent in the contractile phenotype, was decreased by FN. Expressions of matrix Gla and osteopontin, apparent in the synthetic phenotype, were increased by FN. Ang II measured by radioimmunoassay (RIA) was significantly increased in human VSMC by FN. Expression of mRNAs for Ang II-generating proteases cathepsin D, cathepsin G, ACE, and chymase was increased by FN. Expressions of cathepsin D and cathepsin G proteins were also increased by FN. Ang I-generating activity, which was inhibited by an aspartyl protease inhibitor pepstatin A, was readily detected in the conditioned medium from human VSMC. Antisense oligodeoxynucleotides (ODNs) that hybridize with cathepsin D and cathepsin G significantly inhibited FN-increased Ang II in conditioned medium and cell extracts. In VSMC conditioned medium, FN-induced elevation of Ang II was significantly inhibited by temocapril but not by chymostatin. Ang II type 1 receptor antagonist CV11974 completely, and antisense cathepsin D and cathepsin G ODNs partially inhibited the FN-stimulated growth of human VSMC. These results indicate that the change of homogeneous cultures of human VSMC from the contractile to the synthetic phenotype sequentially increases expression of proteases cathepsin D, cathepsin G, and ACE, production of Ang II and productions of growth factors, culminating in VSMC proliferation. These findings implicate a new mechanism for the pathogenesis of human vascular proliferative diseases.     

Knockdown of TRIM27 expression suppresses the dysfunction of mesangial cells in lupus nephritis by FoxO1 pathway

TRIM27 (tripartite motif-containing 27) is a member of the TRIM (tripartite motif) protein family and participates in a variety of biological processes. Some research has reported that TRIM27 was highly expressed in certain kinds of carcinoma cells and tissues and played an important role in the proliferation of carcinoma cells. However, whether TRIM27 takes part in the progression of lupus nephritis (LN) especially in cells proliferation remains unclear. Our study revealed that the overexpression of TRIM27 was observed in the kidneys of patients with LN, lupus mice and mesangial cells exposed to LN plasma which correlated with the proliferation of mesangial cells and ECM (extracellular matrix) deposition. Downregulation of TRIM27 expression suppressed the proliferation of mesangial cells and ECM accumulation in MRL/lpr mice and cultured human mesangial cells (HMCs) by regulating the FoxO1 pathway. Furthermore, the overexpression of FoxO1 remarkably decreased HMCs proliferation level and ECM accumulation in LN plasma-treated HMCs. In addition, the protein kinase B (Akt) signal pathway inhibitor LY294002 significantly reduced the expression of TRIM27 and inhibited the dysfunction of mesangial cells. These above data suggested that TRIM27 mediated abnormal mesangial cell proliferation in kidney of lupus and might be the potential target for treating mesangial cell proliferation of lupus nephritis.     

Expression of genes related to the extracellular matrix in human endothelial cells. Differential modulation by elevated glucose concentrations, phorbol esters, and cAMP.

To identify agents and mechanisms responsible for the thickened basement membranes characteristic of diabetic angiopathy we examined the effects of high glucose (30 mM) on the expression of genes related to extracellular matrix composition and turnover and investigated whether the changes induced by high glucose were mimicked and sustained by activation of protein kinase C or A. In human umbilical vein endothelial cells high glucose increased fibronectin, collagen IV, tissue plasminogen activator (tPA), and plasminogen activator-inhibitor 1 (PAI-1) mRNA levels 2-fold but did not affect type IV and interstitial collagenase expression. Acute treatment with phorbol esters resulted in increased collagen IV, tPA, PAI-1, and interstitial collagenase mRNAs; the type IV collagenase mRNA levels were instead suppressed to 50% of control. Upon longer exposure to phorbol esters (48 h) suppression of fibronectin and PAI-1 mRNAs also occurred. Intracellular elevation of cAMP led to over-expression of fibronectin and type IV collagenase and potentiated the effects of phorbol esters on collagen IV, tPA, and interstitial collagenase expression. The mRNA changes induced by high glucose occurred in the absence of protein kinase C activation or cAMP elevation. These studies indicate that events other than activation of protein kinase C or A bridge high ambient glucose to changes in endothelial cell gene expression that may contribute to diabetic angiopathy.     

A constitutive endogenous osteopontin production is important for macrophage function and differentiation

Macrophages are involved in the pathological process underlying atherosclerosis and constitutively express the multifunctional protein osteopontin which has important exogenous effects on these cells. However, the effect of the endogenous osteopontin expression on macrophage function has been sparsely studied. To shed light on the importance of the endogenous osteopontin expression, RAW 264.7 macrophage-like cells were silenced in osteopontin expression using RNAi. The cells were analysed for basic functions including attachment, migration, apoptosis and for the expression of macrophage differentiation markers and cytokines. The macrophages with silenced osteopontin expression showed impaired migration and an increased rate of serum starvation-induced apoptosis as compared to osteopontin-producing control cells. Furthermore, the cells with silence osteopontin expression had an altered phenotype with monocyte-like characteristics, including decreased expression of macrophage scavenger receptor A type 1. The altered phenotype of these cells could not be reversed by presence of extracellular osteopontin. In addition the cells with silenced osteopontin expression had a lower expression of IL-12 mRNA and the anti-apoptotic Flip mRNA. We conclude that a constitutive endogenous osteopontin production is important for proper basic functions of macrophages and our study indicates that the constitutive osteopontin production is involved in maintaining macrophages in a differentiated phenotype.