Protein kinase D1 overexpression potentiates epidermal growth factor signaling pathway in MCF-7 cells.

BACKGROUND: Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified. METHODS AND RESULTS: By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells. CONCLUSIONS: Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting.

The Role of GH/IGF Axis in Dento-Alveolar Complex from Development to Aging and Therapeutics: A Narrative Review.

The GH/IGF axis is a major regulator of bone formation and resorption and is essential to the achievement of normal skeleton growth and homeostasis. Beyond its key role in bone physiology, the GH/IGF axis has also major pleiotropic endocrine and autocrine/paracrine effects on mineralized tissues throughout life. This article aims to review the literature on GH, IGFs, IGF binding proteins, and their respective receptors in dental tissues, both epithelium (enamel) and mesenchyme (dentin, pulp, and tooth-supporting periodontium). The present review re-examines and refines the expression of the elements of the GH/IGF axis in oral tissues and their in vivo and in vitro mechanisms of action in different mineralizing cell types of the dento-alveolar complex including ameloblasts, odontoblasts, pulp cells, cementoblasts, periodontal ligament cells, and jaw osteoblasts focusing on cell-specific activities. Together, these data emphasize the determinant role of the GH/IGF axis in physiological and pathological development, morphometry, and aging of the teeth, the periodontium, and oral bones in humans, rodents, and other vertebrates. These advancements in oral biology have elicited an enormous interest among investigators to translate the fundamental discoveries on the GH/IGF axis into innovative strategies for targeted oral tissue therapies with local treatments, associated or not with materials, for orthodontics and the repair and regeneration of the dento-alveolar complex and oral bones.

Pyruvate Treatment Restores the Effectiveness of Chemotherapeutic Agents in Human Colon Adenocarcinoma and Pleural Mesothelioma Cells.

Emerging evidence supports the idea that a dysfunction in cell metabolism could sustain a resistant phenotype in cancer cells. As the success of chemotherapeutic agents is often questioned by the occurrence of multidrug resistance (MDR), a multiple cross-resistance towards different anti-cancer drugs represent a major obstacle to cancer treatment. The present study has clarified the involvement of the carbon metabolites in a more aggressive tumor colon adenocarcinoma phenotype and in a chemoresistant mesothelioma, and the role of pyruvate treatment in the reversion of the potentially related resistance. For the first time, we have shown that human colon adenocarcinoma cells (HT29) and its chemoresistant counterpart (HT29-dx) displayed different carbon metabolism: HT29-dx cells had a higher glucose consumption compared to HT29 cells, whereas human malignant mesothelioma (HMM) cells showed a lower glucose consumption compared to HT29 cells, accompanied by a lower pyruvate production and, consequently, a higher production of lactate. When treated with pyruvate, both HT29-dx and HMM cells exhibited a re-established accumulation of doxorubicin and a lower survival ability, a decreased activity of multidrug resistance protein 1 (MRP1) and a restored mitochondrial respiratory chain function, improving the effectiveness of the chemotherapeutic agents in these resistant cancer cells.

Hyperglycemia Promotes Chemoresistance Through the Reduction of the Mitochondrial DNA Damage, the Bax/Bcl-2 and Bax/Bcl-XL Ratio, and the Cells in Sub-G1 Phase Due to Antitumoral Drugs Induced-Cytotoxicity in Human Colon Adenocarcinoma Cells.

Diabetes and cancer are common, chronic, and potentially fatal diseases that frequently co-exist. Observational studies clearly indicate that the risk of several types of cancer is increased in diabetic patients and a number of cancer types have shown a higher mortality rate in patients with hyperglycemic associated pathologies. This scenario could be due, at least in part, to a lower efficacy of the cancer treatments which needs to be better investigated. Here, we evaluated the effects of a prolonged exposure to high glucose (HG) to the response to chemotherapy on human colon adenocarcinoma HT29 and LOVO cell lines. We observed that hyperglycemia protected against the decreased cell viability and cytotoxicity and preserved from the mitochondrial DNA lesions induced by doxorubicin (DOX) and 5-fluorouracil (5-FU) treatments by lowering ROS production. In HT29 cells the amount of intracellular DOX and its nuclear localization were not modified by HG incubation in terms of Pgp, BCRP, MRP1, 5 and 8 activity and gene expression. On the contrary, in LOVO cells, the amount of intracellular DOX was significantly decreased after a bolus of DOX in HG condition and the expression and activity of MPR1 was increased, suggesting that HG promotes drug chemoresistance in both HT29 and LOVO cells, but in a different way. In both cell types, HG condition prevented the susceptibility to apoptosis by decreasing the ratio Bax/Bcl-2 and Bax/Bcl-XL and diminished the level of cytosolic cytochrome c and the cleavage of full length of PARP induced by DOX and 5-FU. Finally, hyperglycemia reduced cell death by decreasing the cell percentage in sub-G1 peak induced by DOX (via a cell cycle arrest in the G2/M phase) and 5-FU (via a cell cycle arrest in the S phase) in HT29 and LOVO cells. Taken together, our data showed that a prolonged exposure to HG protects human colon adenocarcinoma cells from the cytotoxic effects of two widely used chemotherapeutic drugs, impairing the effectiveness of the chemotherapy itself.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Carbon in intimate contact with quartz reduces the biological activity of crystalline silica dusts.

To evaluate the effect of carbonaceous materials on the pathogenic activity of quartz dusts, mixtures of carbon soot (1 and 10%) and quartz (Min-U-Sil) were prepared and then milled so to attain an intimate association of carbon and the quartz surface. Both cellular and cell-free tests show that carbon associated to quartz completely inhibits the typical free radical generation of quartz dusts (through Fenton activity and homolytic cleavage of a C-H bond) and suppresses the oxidative stress and inflammation induced by quartz alone on MH-S murine macrophage cells (lipid peroxidation, nitric oxide release, and tumor necrosis factor-α synthesis). The cytotoxic response to quartz is also largely reduced. An extremely pure quartz milled with 10% of soot showed inactivating effects on the adverse reactions to quartz similar to Min-U-Sil quartz. None of these effects takes place when the same experiments are carried out with mechanically mixed samples, which suggests that carbon acts not just as a radical quencher but because of its association to the quartz surface.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters. METHODS: HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay. RESULTS: In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids. CONCLUSIONS: MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

Pentosan polysulfate inhibits atherosclerosis in Watanabe heritable hyperlipidemic rabbits: differential modulation of metalloproteinase-2 and -9.

Pentosan polysulfate (PPS), a heparinoid compound essentially devoid of anticoagulant activity, modulates cell growth and decreases inflammation. We investigated the effect of PPS on the progression of established atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits. After severe atherosclerosis developed on an atherogenic diet, WHHL rabbits were treated with oral PPS or tap water for 1 month. The aortic intima-to-media ratio and macrophage infiltration were reduced, plaque collagen content was increased, and plaque fibrous caps were preserved by PPS treatment. Plasma lipid levels and post-heparin hepatic lipase activity remained unchanged. However, net collagenolytic activity in aortic extracts was decreased, and the levels of matrix metalloproteinase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP) activity were increased by PPS. Moreover, PPS treatment decreased tumor necrosis factor α (TNFα)-stimulated proinflammatory responses, in particular activation of nuclear factor-κB and p38, and activation of MMPs in macrophages. In conclusion, oral PPS treatment prevents progression of established atherosclerosis in WHHL rabbits. This effect may be partially mediated by increased MMP-2 and TIMP activities in the aortic wall and reduced TNFα-stimulated inflammation and MMP activation in macrophages. Thus, PPS may be a useful agent in inhibiting the progression of atherosclerosis.

Antioxidants prevent the RhoA inhibition evoked by crocidolite asbestos in human mesothelial and mesothelioma cells.

Asbestos is a naturally occurring fibrous silicate, whose inhalation is highly related to the risk of developing malignant mesothelioma (MM), and crocidolite is one of its most oncogenic types. The mechanism by which asbestos may cause MM is unclear. We have previously observed that crocidolite in human MM (HMM) cells induces NF-κB activation and stimulates the synthesis of nitric oxide by inhibiting the RhoA signaling pathway. In primary human mesothelial cells (HMCs) and HMM cells exposed to crocidolite asbestos, coincubated or not with antioxidants, we evaluated cytotoxicity and oxidative stress induction (lipid peroxidation) and the effect of asbestos on the RhoA signaling pathway (RhoA GTP binding, Rho kinase activity, RhoA prenylation, hydroxy-3-methylglutharyl-CoA reductase activity). In this paper we show that the reactive oxygen species generated by the incubation of crocidolite with primary HMCs and three HMM cell lines mediate the inhibition of 3-hydroxy-3-methylglutharyl-CoA reductase (HMGCR). The coincubation of HMCs and HMM cells with crocidolite together with antioxidants, such as Tempol, Mn-porphyrin, and the association of superoxide dismutase and catalase, prevented the cytotoxicity and lipoperoxidation caused by crocidolite alone as well as the decrease of HMGCR activity and restored the RhoA/RhoA-dependent kinase activity and the RhoA prenylation. The same effect was observed when the oxidizing agent menadione was administrated to the cells in place of crocidolite. Such a mechanism could at least partly explain the effects exerted by crocidolite fibers in mesothelial cells.

Estrogens and progression of diabetic kidney damage.

It is generally accepted that estrogens affect and modulate the development and progression of chronic kidney diseases (CKD) not related to diabetes. Clinical studies have indeed demonstrated that the severity and rate of progression of renal damage tends to be greater among men, compared with women. Experimental studies also support the notion that female sex is protective and male sex permissive, for the development of CKD in non-diabetics, through the opposing actions of estrogens and testosterone. However, when we consider diabetes-induced kidney damage, in the setting of either type 1 or type 2 diabetes, the contribution of gender to the progression of renal disease is somewhat uncertain. Previous studies on the effects of estrogens in the pathogenesis of progressive kidney damage have primarily focused on mesangial cells. More recently, data on the effects of estrogens on podocytes, the cell type whose role may include initiation of progressive diabetic renal disease, became available. The aim of this review will be to summarize the main clinical and experimental data on the effects of estrogens on the progression of diabetes-induced kidney injury. In particular, we will highlight the possible biological effects of estrogens on podocytes, especially considering those critical for the pathogenesis of diabetic kidney damage.

Nitric oxide and P-glycoprotein modulate the phagocytosis of colon cancer cells.

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.

Testosterone and 17ß-estradiol have opposite effects on podocyte apoptosis that precedes glomerulosclerosis in female estrogen receptor knockout mice.

Podocyte damage and apoptosis are thought to be important if not essential in the development of glomerulosclerosis. Female estrogen receptor knockout mice develop glomerulosclerosis at 9 months of age due to excessive ovarian testosterone production and secretion. Here, we studied the pathogenesis of glomerulosclerosis in this mouse model to determine whether testosterone and/or 17ß-estradiol directly affect the function and survival of podocytes. Glomerulosclerosis in these mice was associated with the expression of desmin and the loss of nephrin, markers of podocyte damage and apoptosis. Ovariectomy preserved the function and survival of podocytes by eliminating the source of endogenous testosterone production. In contrast, testosterone supplementation induced podocyte apoptosis in ovariectomized wild-type mice. Importantly, podocytes express functional androgen and estrogen receptors, which, upon stimulation by their respective ligands, have opposing effects. Testosterone induced podocyte apoptosis in vitro by androgen receptor activation, but independent of the TGF-ß1 signaling pathway. Pretreatment with 17ß-estradiol prevented testosterone-induced podocyte apoptosis, an estrogen receptor-dependent effect mediated by activation of the ERK signaling pathway, and protected podocytes from TGF-ß1- or TNF-α-induced apoptosis. Thus, podocytes are target cells for testosterone and 17ß-estradiol. These hormones modulate podocyte damage and apoptosis.

17 beta-estradiol and tamoxifen upregulate estrogen receptor beta expression and control podocyte signaling pathways in a model of type 2 diabetes.

Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

RhoA silencing reverts the resistance to doxorubicin in human colon cancer cells.

The efficacy of doxorubicin in the treatment of cancer is limited by its side effects and by the onset of drug resistance. Reverting such resistance could allow the decrease of the dose necessary to eradicate the tumor, thus diminishing the toxicity of the drug. We transfected doxorubicin-sensitive (HT29) and doxorubicin-resistant (HT29-dx) human colon cancer cells with RhoA small interfering RNA. The subsequent decrease of RhoA protein was associated with the increased sensitivity to doxorubicin in HT29 cells and the complete reversion of doxorubicin resistance in HT29-dx cells. RhoA silencing increased the activation of the nuclear factor-kappaB pathway, inducing the transcription and the activity of nitric oxide synthase. This led to the tyrosine nitration of the multidrug resistance protein 3 transporter (MRP3) and contributed to a reduced doxorubicin efflux. Moreover, RhoA silencing decreased the ATPase activity of P-glycoprotein (Pgp) in HT29 and HT29-dx cells as a consequence of the reduced expression of Pgp. RhoA silencing, by acting as an upstream controller of both MRP3 nitration and Pgp expression, was effective to revert the toxicity and accumulation of doxorubicin in both HT29 and HT29-dx cells. Therefore, we suggest that inactivating RhoA has potential clinical applications and might in the future become part of a gene therapy protocol.

Statins-mediated inhibition of rho GTPases as a potential tool in anti-tumor therapy.

Rho GTPases, which control processes such as cell proliferation and cytoskeleton remodeling, are often hyper-expressed in tumors. Several members, such as RhoA/B/C, must be isoprenylated to interact with their effectors. Statins, by inhibiting the synthesis of prenyl groups, may affect RhoA/B/C activity and represent a promising tool in anticancer therapy.

Activation of nuclear factor-kappa B pathway by simvastatin and RhoA silencing increases doxorubicin cytotoxicity in human colon cancer HT29 cells.

Doxorubicin efficacy in cancer therapy is hampered by the dose-dependent side effects, which may be overcome by reducing the drug's dose and increasing its efficacy. In the present work, we suggest that the activation of the nuclear factor-kappaB (NF-kappaB) pathway and of nitric-oxide (NO) synthase increases the doxorubicin efficacy in human colon cancer HT29 cells. To induce NF-kappaB, we took into account the effect of doxorubicin itself and of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin; as NF-kappaB inhibitors, we chose the sesquiterpene lactones parthenolide and artemisinin. Simvastatin increased the NF-kappaB activity and NO synthesis, elicited the tyrosine nitration of the multidrug resistance-related protein 3, and enhanced the doxorubicin intracellular accumulation and cytotoxicity. Simvastatin potentiated the effect of doxorubicin on the NF-kappaB pathway and the inducible NO synthase expression. The effects of simvastatin were due to the inhibition of the small G-protein RhoA and of its effector Rho kinase. Parthenolide and artemisinin prevented all of the statin effects by inducing RhoA/Rho kinase activation. On the other hand, they did not reduce the NF-kappaB translocation and doxorubicin intracellular content when RhoA was silenced by small interfering RNA (siRNA). It is interesting that RhoA siRNA was sufficient to increase NF-kappaB translocation, NO synthase activity, doxorubicin accumulation, and cytotoxicity also in non-stimulated cells. Our results suggest that artemisinin, a widely used antimalarial drug, may impair the response to doxorubicin in colon cancer cells; on the contrary, simvastatin and RhoA siRNA may represent future therapeutic approaches to improve doxorubicin efficacy, reducing the risk of doxorubicin-dependent adverse effects.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress.

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-kappa B and decreased intracellular level of its inhibitor IkB alpha. These effects, accompanied by increased production of H2O2, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-kappa B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

The proangiogenic phenotype of tumor-derived endothelial cells is reverted by the overexpression of platelet-activating factor acetylhydrolase.

PURPOSE: We previously reported that human tumor-derived endothelial cells (TEC) have an angiogenic phenotype related to the autocrine production of several angiogenic factors. The purpose of the present study was to evaluate whether an enhanced synthesis of platelet-activating factor (PAF) might contribute to the proangiogenic characteristics of TEC and whether its inactivation might inhibit angiogenesis. EXPERIMENTAL DESIGN: To address the potential role of PAF in the proangiogenic characteristics of TEC, we engineered TEC to stably overexpress human plasma PAF-acetylhydrolase (PAF-AH), the major PAF-inactivating enzyme, and we evaluated in vitro and in vivo angiogenesis. RESULTS: TECs were able to synthesize a significantly enhanced amount of PAF compared with normal human microvascular endothelial cells when stimulated with thrombin, vascular endothelial growth factor, or soluble CD154. Transfection of TEC with PAF-AH (TEC-PAF-AH) significantly inhibited apoptosis resistance and spontaneous motility of TEC. In addition, PAF and vascular endothelial growth factor stimulation enhanced the motility and adhesion of TEC but not of TEC-PAF-AH. In vitro, TEC-PAF-AH lost the characteristic ability of TEC to form vessel-like structures when plated on Matrigel. Finally, when cells were injected s.c. within Matrigel in severe combined immunodeficiency mice or coimplanted with a renal carcinoma cell line, the overexpression of PAF-AH induced a significant reduction of functional vessel formation. CONCLUSIONS: These results suggest that inactivation of PAF, produced by TEC, by the overexpression of plasma PAF-AH affects survival, migration, and the angiogenic response of TEC both in vitro and in vivo.

HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.

OBJECTIVE: To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). DESIGN: Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. METHODS: Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. RESULTS: We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. CONCLUSION: The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

Calpain activation and secretion promote glomerular injury in experimental glomerulonephritis: evidence from calpastatin-transgenic mice.

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.