TSP-1 Expression Research Articles

Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature

Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter‐Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1‐31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum. © 2015 Wiley Periodicals, Inc.

Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts.

Sustained endothelial expression of HoxA5 in vivo impairs pathological angiogenesis and tumor progression.

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.

Interaction of thrombospondin1 and CD36 contributes to obesity-associated podocytopathy

Obesity is associated with podocyte injury and the development of proteinuria. Elevated plasma free fatty acid is one of the characteristics of obesity and has been linked to podocyte dysfunction. However, the mechanisms remain unclear. In the current study, we examined the effect of saturated free fatty acid (FFA) on human podocyte apoptosis and function in vitro. The mechanism and its in vivo relevance were also determined. We found that FFA treatment induced human podocyte apoptosis and dysfunction, which was associated with increased expression of a matricellular protein-thrombospondin1 (TSP1). FFA stimulated TSP1 expression in podocytes at the transcriptional levels through activation of MAPK pathway. Addition of purified TSP1 to cell culture media induced podocyte apoptosis and dysfunction. Tis effect is though a TGF-β independent mechanism. Moreover, peptide treatment to block TSP1 binding to its receptor-CD36 attenuated FFA induced podocyte apoptosis, suggesting that TSP1/CD36 interaction mediates FFA-induced podocyte apoptosis. Importantly, using a diet-induced obese mouse model, in vivo data demonstrated that obesity-associated podocyte apoptosis and dysfunction were attenuated in TSP1 deficient mice as well as in CD36 deficient mice. Taken together, these studies provide novel evidence that the interaction of TSP1 with its receptor CD36 contributes to obesity — associated podocytopathy.

Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature.

Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

Synergistic induction of CCL2/MCP-1 expression driven by oncostatin M and IL-1β in human proximal tubular cells depends on STAT3 and p65 NFκB/RelA.

In response to tubular injury, production, and secretion of cytokines, chemokines or extracellular matrix components by human proximal tubular epithelial cells (PTC) directly contribute to the development of tubulointerstitial inflammation and fibrosis. Here, we report a novel stimulatory and synergistic effect of oncostatin M (OSM) on proinflammatory CCL2/MCP-1 mRNA expression in human PTC. Although OSM inhibited IL-1β- and TNF-α-mediated mRNA expression of matricellular proteins TSP-1 and tenascin C (TNC), it acted synergistically with these two proinflammatory cytokines to induce CCL2 mRNA expression for up to 24 h. Stimulation of two independent human PTC lines with OSM alone led to a rapid and strong induction of this chemokine within the first hour of ligand administration, which subsequently returned toward basal levels in between 3 and 24 h and finally switched into a significant OSM-mediated 70% inhibition of basal CCL2 mRNA expression after 48 h of incubation. In contrast to OSM, which stimulated both STAT1/3 and ERK1/2 signaling, IL-1β led to a strong phosphorylation of p65 NFκB/RelA, SMAD2/3, and p38 MAPK in human PTC. Selective silencing of these signaling molecules revealed that p65 NFκB/RelA is involved in IL-1β-mediated stimulation of CCL2 mRNA, and that superinduction of CCL2 mRNA expression in the presence of both OSM and IL-1β at least partially depends on STAT3 signaling. Thus, with respect to the expression of the proinflammatory chemokine CCL2, OSM may stimulate acute inflammation via its synergistic effect with other proinflammatory cytokines early after injury.

Association Between a Pro-Inflammatory Axis Comprised By TSP1-TGFβ-CTGF with Three microRNAs, Mir-19a, miR122 and Mir-133a in the Pathophysiology of Diabetic Retinopathy: A Therapeutic Novel Modality

Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication.Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark.Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p<0.005), miR-19a (p<0.008), miR-122 (p<0.02) or PDR (decreased), miR-19a p<0.000096) and miR-122 (p<0.005). miR19a has been implicated with expression of TSP1 and CTGF, glucose levels, insulin resistance and diabetes-associated pancreatic cancer. miR-133a has been associated with cardiac hyperthrophy, fibrosis and heart failure in diabetes. miR-122 has been identified as a potential biomarker for non-alcoholic fatty liver. In summary our results link for the first time in the literature three circulating microRNAs with a the above axis in the pathophysiology of NPDR and PDR. DisclosuresNo relevant conflicts of interest to declare.

Adenosine stimulates angiogenesis by up-regulating production of thrombospondin-1 by macrophages.

Increase of blood capillary density at the interface between normal and ischemic tissue after acute MI reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component TSP-1, but its role in angiogenesis is not clear, as both anti- and proangiogenic properties have been reported. It is unknown whether TSP-1 is modulated by other factors released during cardiac injury. Among these, Ado is a well-known promoter of angiogenesis. This study determined whether Ado modulates TSP-1 expression and the implication on angiogenesis. Ado dose dependently increased the production of TSP-1 by human macrophages. With the use of agonists and antagonists of AdoRs, coupled to RNA interference, we observed that this effect is mediated via A2AR and A2BR. The Ado effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator) and blocked by the PKA inhibitor H89. Conditioned medium from Ado-treated macrophages stimulated microvessel outgrowth from aortic ring explants by 400%, and induced vessel formation in matrigel plugs. Microvessel outgrowth and vessel formation were blocked completely by addition of anti-TSP-1 antibodies to conditioned medium. Chronic administration of Ado to rats after MI maintained long-term expression of TSP-1 in the infarct border zone, and this was associated with enhanced border-zone vascularization. Ado up-regulates TSP-1 production by macrophages, resulting in stimulation of angiogenesis. The mechanism involves A2AR and A2BR and is mediated through the cAMP/PKA pathway. This information may be important when designing Ado-based therapies of angiogenesis.

Anti-angiogenic pathway associations of the 3p21.3 mapped BLU gene in nasopharyngeal carcinoma.

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70–90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.

Pathological significance and prognostic role of microvessel density, evaluated using CD31, CD34, and CD105 in prostate cancer patients after radical prostatectomy with neoadjuvant therapy.

BACKGROUNDNeoadjuvant hormonal therapy (NHT) is performed to improve the outcome in organ-confined prostate cancer. However, there is little information regarding the relationship between angiogenesis and NHT. The aim of this study was to identify a suitable method to evaluate the angiogenic status of tissue, and to determine the prognostic value of this method for biochemical recurrence in patients who had undergone radical prostatectomy after NHT.METHODSWe analyzed 108 formalin-fixed specimens from patients treated by radical prostatectomy. NHT was administered in 48 patients (52.9%) and 60 patients who had a similar Gleason score and pT stage were selected as a non-NHT treated control group. Microvessel density (MVD) was measured using anti-CD31, anti-CD34, and anti-CD105 antibodies. The expressions of vascular endothelial growth factor (VEGF)-A and thrombospondin (TSP)-1 were also evaluated by immunohistochemistry. The prognostic value of CD31-, CD34-, and CD105-MVD for biochemical recurrence was investigated.RESULTSThe mean/SD of CD105-MVD in the NHT group (13.3/4.7) was significantly (P < 0.001) lower than that in the non-NHT group (125.8/7.3). In the NHT group, CD105-MVD was associated with pT stage and it was positively correlated with VEGF-A expression (r = 0.56, P < 0.001) and negatively correlated with TSP-1 expression (r = 0.42, P = 0.003). CD105-MVD was identified as a significant predictor of biochemical recurrence (BCR) in patients treated with NHT (log rank test, P < 0.001). Although CD31- and CD34-MVD were significantly associated with pT stage or Gleason score in non-NHT group, they were not associated with pathological features and BCR in NHT group.CONCLUSIONSOur results indicate that CD105-MVD reflects the angiogenic conditions in prostate cancer tissues treated with NHT. CD105-MVD was also identified as a significant and independent predictor of biochemical recurrence in prostate cancer patients who underwent radical prostatectomy with NHT. Prostate 75:84–91, 2015. © 2014 The Authors. The Prostate published by Wiley Periodicals, Inc.

MMP-12 deficiency attenuates angiotensin II-induced vascular injury, M2 macrophage accumulation, and skin and heart fibrosis.

MMP-12, a macrophage-secreted elastase, is elevated in fibrotic diseases, including systemic sclerosis (SSc) and correlates with vasculopathy and fibrosis. The goal of this study was to investigate the role of MMP-12 in cardiac and cutaneous fibrosis induced by angiotensin II infusion. Ang II-induced heart and skin fibrosis was accompanied by a marked increase of vascular injury markers, including vWF, Thrombospondin-1 (TSP-1) and MMP-12, as well as increased number of PDGFRβ+ cells. Furthermore Ang II infusion led to an accumulation of macrophages (Mac3+) in the skin and in the perivascular and interstitial fibrotic regions of the heart. However, alternatively activated (Arg 1+) macrophages were mainly present in the Ang II infused mice and were localized to the perivascular heart regions and to the skin, but were not detected in the interstitial heart regions. Elevated expression of MMP-12 was primarily found in macrophages and endothelial cells (CD31+) cells, but MMP-12 was not expressed in the collagen producing cells. MMP-12 deficient mice (MMP12KO) showed markedly reduced expression of vWF, TSP1, and PDGFRβ around vessels and attenuation of dermal fibrosis, as well as the perivascular fibrosis in the heart. However, MMP-12 deficiency did not affect interstitial heart fibrosis, suggesting a heterogeneous nature of the fibrotic response in the heart. Furthermore, MMP-12 deficiency almost completely prevented accumulation of Arg 1+ cells, whereas the number of Mac3+ cells was partially reduced. Moreover production of profibrotic mediators such as PDGFBB, TGFβ1 and pSMAD2 in the skin and perivascular regions of the heart was also inhibited. Together, the results of this study show a close correlation between vascular injury markers, Arg 1+ macrophage accumulation and fibrosis and suggest an important role of MMP-12 in regulating these processes.

ROLE OF PRO- AND ANTIANGIOGENIC FACTORS IN PLACENTAL DEVELOPMENT

Vascular network of placenta exhibits plasticity, and undergoes various dynamic changes in the course of pregnancy. Placental development is controlled by various cytokines and growth factors, and their ratios show strong fluctuations during pregnancy. The purpose of present study was to evaluate expression and secretion of proangiogenic and antiangiogenic factors by placental tissue at early stages (1st trimester) and late terms (3rd trimester) of normally proceeding pregnancy. It was established that at earlier stages (1st trimester), placental tissue produced larger quantities of proangiogenic factors (VEGF, PDGF, IL-8, MMP-2), in comparison to the 3rd trimester of pregnancy. By the 3rd trimester, VEGF-R3 expression and bFGF secretion by placental cells was also decreased, accompanied by increased production of angiogenin. Moreover, a suppressed production of antiangiogenic factors TSP-1 and TGFβ and expression of TGFβ-R1, CD105 receptors by placenta tissue was registered by the 3rd trimester of pregnancy. In permanently evolving placental tissue, an excess of angiogenic factors is observed. Meanwhile, antiangiogenic factors also seem to play an essential role in formation of placental tissue, thus providing angiogenesis inhibition. The study was supported by Presidential grants of Russian Federation N НШ-1066.2008.7, МК-1355.2007.7, and by a grant of Russian Foundation for Basic Research № 070400155. (Med. Immunol., vol. 10, N 4-5, pp 347-352) .

Defective expression of scavenger receptors in celiac disease mucosa.

Celiac disease (CD) is a gluten sensitive enteropathy characterized by a marked infiltration of the mucosa with immune cells, over-production of inflammatory cytokines and epithelial cell damage. The factors/mechanisms that sustain and amplify the ongoing mucosal inflammation in CD are not however fully understood. Here, we have examined whether in CD there is a defective clearance of apoptotic cells/bodies, a phenomenon that helps promote tolerogenic signals thus liming pathogenic responses. Accumulation of apoptotic cells and bodies was more pronounced in the epithelial and lamina propria compartments of active CD patients as compared to inactive CD patients and normal controls. Expression of scavenger receptors, which are involved in the clearance of apoptotic cells/bodies, namely thrombospondin (TSP)-1, CD36 and CD61, was significantly reduced in active CD as compared to inactive CD and normal mucosal samples. Consistently, lamina propria mononuclear cells (LPMC) of active CD patients had diminished ability to phagocyte apoptotic cells. Interleukin (IL)-15, IL-21 and interferon-γ, cytokines over-produced in active CD, inhibited the expression of TSP-1, CD36, and CD61 in normal intestinal LPMC. These results indicate that CD-related inflammation is marked by diminished clearance of apoptotic cells/bodies, thus suggesting a role for such a defect in the ongoing mucosal inflammation in this disorder.

Expression of circulating vascular endothelial growth factor-antagonizing cytokines and vascular stabilizing factors prior to and following bypass surgery in patients with moyamoya disease.

The aim of the present study was to investigate the levels of vascular endothelial growth factor (VEGF)-antagonizing cytokines and VEGF-influenced vascular stabilizing cytokines in patients with moyamoya disease (MMD) and the association with postoperative collateral vessel formation. The study population included 53 MMD patients that had undergone indirect bypass surgery and 50 healthy controls. Serum levels of VEGF, thrombospondin-1 (TSP-1), TSP-2, soluble VEGF receptor-1 (sVEGFR-1), sVEGFR-2, endostatin, angiopoietin-1 (Ang-1) and Ang-2 were measured at the baseline (preoperative) and at day seven following surgery. Postoperative collateralization assessment was conducted upon the six-month follow-up cerebral angiography. Cytokine levels were compared between patients with good or poor collateral formation. Compared with the healthy controls, MMD patients exhibited lower baseline levels of sVEGFR-1 (P<0.0001) and sVEGFR-2 (P<0.0001), but higher VEGF expression (P<0.0001). Ang-1 and Ang-2 levels did not exhibit any difference between the two groups. On day seven following surgery, MMD patients exhibited an almost unchanged sVEGFR-1 and sVEGFR-2 expression level, but upregulated expression of VEGF (P<0.0001), Ang-1 (P<0.0001) and TSP-2 (P<0.0001). The six-month follow-up angiographies revealed that 21 patients (45.65%) that had undergone the same surgical procedure achieved good collateralization. Patients with good collateral formation appeared to have lower sVEGFR-1 and sVEGFR-2 levels prior to (P=0.029 and P=0.045, respectively) and at day seven (P=0.044 and P=0.047, respectively) following bypass surgery when compared with the patients with worse collateralization. Therefore, sVEGFR-1 and sVEGFR-2 may play a role in the pathogenesis of MMD. Lower levels of sVEGFR-1 and sVEGFR-2 indicated better postoperative collateralization in the six months following indirect bypass surgery. However, Ang-1 and Ang-2 may not be specifically involved in the course of MMD.

Acute and Chronic Mu Opioids Differentially Regulate Thrombospondins 1 and 2 Isoforms in Astrocytes

Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but by distinct mechanisms.

Calcitriol reduces thrombospondin-1 and increases vascular endothelial growth factor in breast cancer cells: Implications for tumor angiogenesis

Calcitriol, a potent antineoplastic vitamin D metabolite, inhibits proliferation, induces apoptosis and slows the growth of tumors. Calcitriol also may exert either antiangiogenic or proangiogenic effects depending on the tissue. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (Tsp-1) are key factors involved in promoting and inhibiting angiogenesis, respectively. The effects of calcitriol on Tsp-1 have not been studied in the mammary gland, while VEGF regulation is not clear, since opposite outcomes have been demonstrated. Therefore, the present study was undertaken to investigate the effects of calcitriol on VEGF and Tsp-1 expression in primary breast tumor-derived cells and a panel of established breast cancer cell lines. In vivo studies in athymic mice were also performed in order to gain further insight into the biological effects of calcitriol on angiogenesis.Real time-PCR and ELISA analyses showed that calcitriol stimulated VEGF mRNA expression and protein secretion while elicited the opposite effect on Tsp-1 in 7 out of 8 cell lines studied, independently of the cell phenotype (P<0.05 in n=5). In vivo, calcitriol significantly inhibited the relative tumoral volume after 4 weeks of treatment; however, serum VEGF was higher in calcitriol-treated animals compared to controls (P<0.05). The integrated fluorescence intensity analysis of CD31, a vessel marker, showed that xenografted breast cancer cells developed tumors with similar vascular density regardless of the treatment. Nevertheless, larger necrotic areas were observed in the tumors of calcitriol-treated mice compared to controls. Since the antineoplastic activity of calcitriol has been consistently demonstrated in several studies including this one, our results suggest that the antitumoral effect of calcitriol in vivo involve different mechanisms not necessarily related to the inhibition of tumor vascularization. Overall, our findings indicate that calcitriol can impact the angiogenic process in breast cancer by regulating VEGF and Tsp-1 expression.This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

Non-small cell lung cancer is susceptible to induction of DNA damage responses and inhibition of angiogenesis by telomere overhang oligonucleotides

Exposure of the telomere overhang acts as a DNA damage signal, and exogenous administration of an 11-base oligonucleotide homologous to the 3′-telomere overhang sequence (T-oligo) mimics the effects of overhang exposure by inducing senescence and cell death in non-small cell lung cancer (NSCLC) cells, but not in normal bronchial epithelial cells. T-oligo-induced decrease in cellular proliferation in NSCLC is likely directed through both p53 and its homolog, p73, with subsequent induction of senescence and expression of senescence-associated proteins, p21, p33ING, and p27Kip1 both in vivo and in vitro. Additionally, T-oligo decreases tumor size and inhibits angiogenesis through decreased VEGF signaling and increased TSP-1 expression.

Pentacyclic Triterpenes from Terminalia arjuna Show Multiple Benefits on Aged and Dry Skin

Background: Pentacyclic triterpenoids improve epidermal barrier function and induce collagen production. Here, their effects on cutaneous aging by means of objective instrumental measurements were elucidated. Methods: Reconstituted human epidermis, cultivated keratinocytes and fibroblasts were incubated with Terminalia arjuna triterpenes (T. arjuna bark extract), and mRNA and protein expression of various genes was determined using microarray analysis, qRT-PCR and ELISA techniques. Clinical efficacy of T. arjuna bark extract versus vehicle control cream was elucidated in 30 patients and transepidermal water loss (TEWL), skin hydration and elasticity were measured. Another 30 female patients in their postmenopausal phase were treated with a similar regime, and skin sebum content, cutaneous blood microcirculation and skin density/echogenicity were assessed. Results: Incubation with T. arjuna triterpenes increased FGF-2, TSP-1, TGF-β and CTGF expression, and VEGF secretion in vitro. Elevated lactate dehydrogenase release upon sodium dodecyl sulphate challenge was reversed by the application of T. arjuna bark extract. T. arjuna bark extract decreased TEWL, improved skin moisturization, reduced scaliness and led to significantly improved skin elasticity. Also, increases in blood microflow and skin sebum content as well as improved skin thickness/echogenicity were noted on postmenopausal skin, resulting in visible reduction of sagging skin on the jowls as demonstrated by digital photography. Conclusion:T. arjuna bark extract appears as an innovative active ingredient that exerts versatile antiaging properties in vitro and in vivo.

Tumor Necrosis Factor Inhibition Modulates Thrombospondin-1 Expression in Human Inflammatory Joint Disease through Altered NR4A2 Activity

We examined thrombospondin-1 (THBS1, alias TSP-1) expression in human synovial tissue (ST) during the resolution phase of chronic inflammation and elucidated its transcriptional regulation by the orphan receptor 4A2 (NR4A2). In vivo, rheumatoid arthritis (RA) serum and ST revealed altered expression levels and tissue distribution of TSP-1. After anti-tumor necrosis factor therapy, a reciprocal relationship between TSP-1 and NR4A2 expression levels was measured in patients with clinical and ST responses to biological treatment. In vitro, primary RA fibroblast-like synoviocytes (FLSs) expressed minimal TSP-1 mRNA levels with high transcript levels of NR4A2, vascular endothelial growth factor (VEGF), and IL-8 measured. Hypoxic modulation of RA FLSs resulted in inverse expression levels of TSP-1 compared with NR4A2, IL-8, and VEGF. Ectopic NR4A2 expression led to reduced TSP-1 mRNA and protein levels with concomitant increases in proangiogenic mediators. NR4A2 transcriptional activity, independent of DNA binding, repressed the hTSP-1 promoter leading to reduced mRNA and protein release in immortalized K4IM FLSs. Bioinformatic and deletion studies identified a 5' region of the TSP-1 promoter repressed by NR4A2 and proangiogenic transcription factors, including NF-κB and Ets1/2. Stable depletion of NR4A2 levels resulted in a shift in the TSP-1/VEGF expression ratio. Thus, modulation of TSP-1 expression is achieved through anti-tumor necrosis factor therapy effects on specific transcriptional networks, suggesting that enhanced TSP-1 expression may help restore tissue homeostasis during resolution of inflammation.

Human cytomegalovirus infection modulates thrombospondins 1 and 2 in primary fetal astrocytes

Transmission of human cytomegalovirus (HCMV) to the fetus is the most common type of intrauterine infection; the mechanism of HCMV pathogenesis in the developing central nervous system remains unclear. Thrombospondins 1 and 2 (TSP1, TSP2) produced by immature astrocytes are critical for fetal synaptogenesis. To examine the effect of HCMV on fetal astrocytes, human fetal astrocytes were isolated and cultured with HCMV AD169. Cells were harvested at different time points. Protein and mRNA expressions of TSP1 and TSP2 were determined using RT-qPCR, western blotting analysis, and enzyme-linked immunosorbent assay. The results showed that HCMV infection induced time-dependent decreases in mRNA and protein expressions of both TSP1 and TSP2 in astrocytes. Flow cytometry was used to detect apoptosis of HCMV-infected astrocytes, and the result indicated that there was no linkage between cell apoptosis and the decrease in TSP1 and TSP2 expressions induced by HCMV infection. When ganciclovir treatment was performed on HCMV-infected astrocytes, results showed that ganciclovir treatment inhibited the reduction of TSP1 and TSP2 expression in astrocytes. In the further study, pEGFP-N3-IE1 was transfected into astrocytes to identify that it was not IE1 but active viral replication that was essential in the continuous decrease of TSP1 and TSP2 expressions in HCMV-infected astrocytes.