Regulation Of Extracellular Matrix Remodeling Research Articles

PF127/bleomycin hydrogel promotes subcutaneous extracellular matrix remodeling and fibrosis to construct personalized flaps through the TGFβ-Col signaling pathway

Background Skin flap transplantation is used to effectively reconstruct defects of the hand and foot skin and soft tissues. We here investigated the effect of the PF127/bleomycin (BLM) hydrogel on the extracellular matrix (ECM) remodeling of skin flaps and the underlying mechanism, thereby providing a new reference point for personalized flap modification and overcoming abrasion resistance- and stability-associated difficulties. Methods The appropriate PF127/BLM concentration was selected based on the gelation time and drug release curve. Migration assays, scratch assays, and live/dead staining were conducted to verify the effect of PF127/BLM on human skin fibroblasts (HSFs). The effects of PF127/BLM on the ECM were assessed through hematoxylin and eosin and Masson staining. Additionally, we examined the expression of ECM remodeling-related genes and proteins involved in their associated signaling pathway. Finally, the effects of PF127/BLM on organ fibrosis and toxicity to liver and kidney functions were assessed in mice. Results A 25% PF127/BLM hydrogel was selected as the study concentration. PF127/BLM augmented HSF chemotaxis and proliferation. Furthermore, PF127/BLM promoted subcutaneous ECM remodeling and fibrosis, increased the flap dermis thickness, and reduced the toxic side effects of BLM on liver/lung fibrosis and liver/kidney function. Additional studies confirmed that the PF127/BLM-mediated regulation of ECM remodeling in skin flaps was associated with TGFβ-Col signaling pathway activation. Conclusion The PF127/BLM hydrogel promoted subcutaneous ECM remodeling and fibrosis, which aided the construction of personalized flaps through the TGFβ-Col signaling pathway, with decreased hepatic, pulmonary, and renal toxicities.

TRANSCRIPTOMIC ANALYSIS OF URINARY EXTRACELLULAR VESICLES IN HYPERTENSIVE PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: To perform transcriptomic analysis of RNA samples extracted from urinary extracellular vesicles (uEVs) of patients with primary aldosteronism (PA), subtype aldosterone-producing adenoma (APA), before and after adrenalectomy, and patients with essential hypertension (EH). Then, to compare the differentially expressed genes (DEGs) for evaluating gene expression differences between PA and EH, and PA before and after adrenalectomy. Design and method: Urinary extracellular vesicles were isolated using a commercially available precipitating reagent and differential centrifugations. RNA libraries were prepared according to the TruSeq Stranded Total RNA protocol and sequenced by NextSeq System (Illumina). Results: Gene set enrichment analysis on DEGs in 5 patients with PA compared to 8 EH subjects revealed several significantly overrepresented classes of transcripts, including ion binding, sodium ion transmembrane transport and collagen-containing extracellular matrix. Among the more interesting genes, we found some associated to the activity of voltage-dependent ion channels (CACNA2D1, CACNA2D4, CACNG2, SCN3A, SCN5A, SCN9A, SCN11A). Moreover, in order to identify potentially aldosterone-regulated genes, we focused on DEGs in common between the comparisons APA pre-surgery vs EH and APA pre surgery vs APA post-surgery. We found, among DEGs shared across these two groups, some transcripts involved in the regulation of extracellular matrix remodeling and Phosphoinositide 3-kinases pathway (CST3, EPHB2, ITGA5, PIK3R2). Conclusions: This study contributes to a more precise characterization of the molecular mechanisms underlying PA. To our knowledge, this is the first transcriptomic study on uEVs from patients with PA and EH, confirming evidence found in other studies on different tissues (biological fluids and adrenal tissue). Our results suggest that the mRNA content of these vesicles may reflect the pathophysiological processes occurring in the urinary tract and especially in the kidney.

Substrate Stiffness-Driven Membrane Tension Modulates Vesicular Trafficking via Caveolin-1.

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the β1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.

Crosstalk between YAP and TGFβ regulates SERPINE1 expression in mesenchymal lung cancer cells.

Serpin familyE member1(SERPINE1), a serine proteinase inhibitor, serves as an important regulator of extracellular matrix remodeling. Emerging evidence suggests that SERPINE1 has diverse roles in cancer and is associated with poor prognosis. However, the mechanism via which SERPINE1 is induced in cancer has not been fully determined. In order to examine the molecular mechanism of SERPINE1 expression, the present study took advantage of the isogenic pair of lung cancer cells with epithelial or mesenchymal features. Using genetic perturbation and following biochemical analysis, the present study demonstrated that SERPINE1 expression was upregulated in mesenchymal lung cancer cells and promoted cellular invasiveness. Yes‑associated protein (YAP)‑dependent SERPINE1 expression was modulated by treatment with a Rho‑associatedproteinkinase inhibitor, Y27632. Moreover, TGFβ treatment supported YAP‑dependent SERPINE1 expression, and an enhanced TGFβ response in mesenchymal lung cancer cells promoted SERPINE1 expression. TGFβ‑mediated SERPINE1 expression was significantly attenuated by knockdown of YAP or transcriptional co‑activator with PDZ‑binding motif, suggesting that crosstalk between the TGFβ and YAP pathways underlies SERPINE1 expression in mesenchymal cancer cells.

CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells

Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified “late domain” motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.

Calcification and Thrombosis as Mediators of Bioprosthetic Valve Deterioration

Calcific aortic valve disease is the most common indication for surgical aortic valve replacement (SAVR) and transcatheter aortic valve replacement (TAVR) of diseased valves worldwide. 1 For years,...

2266. Persistent Inflammation in HIV Patients With Community-Acquired Pneumonia and Its Correlation With Lung Injury

BackgroundHIV patients face higher rates of morbidity compared with the general population, largely due to the earlier development of age related diseases (cardiovascular, kidney, and liver disease). While it is likely that chronic immune activation and inflammation are the main contributors to this process, it’s relation to lung injury in HIV remains unknown. Despite restoration of systemic immune function following Antiretroviral Therapy, the risk for lower respiratory tract infection remain elevated in the HIV population. The objective of the study was to assess the relationship between pulmonary inflammation and lung injury.MethodsA prospective cohort study was performed, participants include patients hospitalized in Hospital Universitario San Vicente Fundación and Clínica SOMA, in Colombia. Patients were eligible if they were over the age of 18 and had a documented HIV infection or if they have HIV with newly diagnosed community acquired pneumonia (CAP). The main exclusion criteria were chronic lung disease and immunosuppression that is not due to HIV. Patients belonged to two groups: HIV and HIV + CAP. Plasma, sputum samples and pulmonary function test measurements (PFT) were retrieved within 48 hours of hospital admission and at one month follow-up. The concentrations of 13 biomarkers were measured and correlated with PFT values, followed by a comparison between the two groups.ResultsPrinciple Component Analysis revealed that CCL3, CCL4, BAFF, APRIL, and TIMP-1 accounts for the majority of the variation between the two groups. Furthermore, Kruskal–Wallis testing demonstrates that BAFF and CCL3 are elevated in the HIV + CAP group, compared with the HIV group (P < 0.005). Other markers of bacterial translocation and monocyte activation did not differ between these groups. FVC and FEV1 measurements are lower in the HIV + CAP group compared with the HIV group, while FEV1/FVC remain constant.ConclusionThe results of this study identify a unique constellation of biomarkers in HIV patients with CAP, this constellation of biomarkers consists of pro-inflammatory cytokines and regulators of extracellular matrix remodeling, hinting at the occurrence of an inflammatory and tissue injuring process in the lungs. This is supported by the restrictive ventilation pattern seen in this group of patients.Disclosures All authors: No reported disclosures.

Myh10 deficiency leads to defective extracellular matrix remodeling and pulmonary disease

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.

Plasma matrix metalloproteinases are associated with incident cardiovascular disease and all-cause mortality in patients with type 1 diabetes: a 12-year follow-up study

BackgroundAltered regulation of extracellular matrix remodeling by matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) may contribute to vascular complications in type 1 diabetes. We investigated associations between plasma MMP-1, -2, -3, -9, -10 and TIMP-1, and cardiovascular events and all-cause mortality in type 1 diabetic patients.MethodsWe prospectively followed 337 type 1 diabetic patients [mean age 41.4 years (9.6), 39% female], 170 with and 167 without diabetic nephropathy, with median follow-up of 12.3 years. Survival analyses were applied to investigate differences in plasma MMP-1, -2, -3, -9, -10, and TIMP-1-levels in patients with and without a cardiovascular event and in those who died vs survivors. All analyses were adjusted for age, sex, duration of diabetes, HbA1c, nephropathy and for other conventional cardiovascular risk factors.ResultsAfter adjustment for potential confounders, higher MMP-2 plasma levels were significantly associated with higher incidence of cardiovascular events [HR 1.49 (95% CI 1.11; 1.99)], and higher plasma levels of MMP-1 [1.38 (1.07; 1.78)], MMP-2 [1.60 (1.19; 2.15)] and MMP-3 [1.39 (1.05; 1.85)] were associated with all-cause mortality. All associations were independent of low-grade inflammation and endothelial dysfunction as estimated by plasma markers. Associations between MMP-2 and cardiovascular events and between MMP-3 and mortality were attenuated after further adjustment for eGFR and changes in eGFR.ConclusionsHigher levels of MMP-2 are associated with CVD and higher MMP-1, -2 and -3 with all-cause mortality. In addition, associations between MMP-2 and CVD, and MMP-3 and mortality were attenuated after adjustment for eGFR while both MMPs were associated with eGFR decline, indicating a possible mediating role of eGFR.

Mesenchymal stem cells in skin wound healing

The integrity of healthy skin plays a crucial role in maintaining physiological homeostasis of the human body. Chronic conditions such as diabetes mellitus or peripheral vascular diseases can lead to impaired wound healing. Skin wound healing purposes focusing on the main phases of wound healing, i.e., inflammation, proliferation, epithelialization, angiogenesis, remodeling, and scarring. This is a complex process, which is dependent on many cell types and mediators interacting in a highly sophisticated temporal sequence. Although some interactions during the healing process are crucial, redundancy is high and other cells or mediators can adopt functions or signaling without major complications. Mesenchymal stem cells have an alternative role due to special properties such as the capacity for self-renewal and multi-lineage differentiation, immunomodulatory effect, alleviation of inflammatory response, induction of angiogenesis, regulation of extracellular matrix remodeling, excellent migration and secretion of growth factors and cytokines in wound healing. We summarized current research on the mechanisms of mesenchymal stem cells with their isolation, specific markers, differentiation capacity, and the functional activities to evaluate wound healing application.

Differential expression of Low density lipoprotein Receptor–related Protein 1 (LRP-1) and matrix metalloproteinase-9 (MMP-9) in prostate gland: From normal to malignant lesions

BackgroundMetalloproteinases (MMPs) are relevant modulators of inflammation, tumor microenvironment, cancer invasion and metastasis. They can be regulated by the Low density lipoprotein Receptor–related Protein 1 (LRP-1), a receptor reported to mediate the clearance of lipoproteins, extracellular matrix (ECM) macromolecules and proteinases. The aim of this study was to evaluate the expression of LRP-1, MMP-2 and MMP-9 across various grades of prostatic diseases as benign prostatic hyperplasia (BPH), BPH plus prostatitis (BPH+P), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PCa). MethodsLRP-1 was analyzed using immunohistochemistry and MMPs proteolytic activity by zymography in prostate tissues with different prostatic diseases. ResultsLRP-1 was detected in epithelial cells in BPH (16/18), BPH+P (21/21) and HGPIN (6/6), with a staining intensity of 1+, 1+/2+ and 3+, respectively. In PCa, LRP-1 was absent in 19/27 samples while a low expression was observed in 8/27 biopsies. MMP-9 activity was higher and statistically significant in PCa than in BPH (p≤0.01). ConclusionConsidering that LRP-1, by mediating the clearance of MMPs, is involved in the regulation of ECM remodeling and cell migration, we conclude that a decreased expression of LRP-1 could be involved with the increasing activity of MMPs shown in cancers.

Sex-based difference in Achilles peritendinous levels of matrix metalloproteinases and growth factors after acute resistance exercise.

Several recent investigations have demonstrated that the ability of various tendons to alter structural and functional properties in response to exercise are muted in women compared with men. We hypothesize that this disparity between men and women may be due to a reduced tendon production of key mediators of tendon extracellular matrix (ECM) remodeling in response to mechanical loading, e.g., exercise. Using microdialysis before and after an acute bout of resistance exercise, we evaluated Achilles peritendinous levels of insulin-like growth factor-1 (IGF-1) and interleukin-6 (IL-6), which have both been shown to increase tendon collagen synthesis. Additionally, the matrix remodeling enzymes matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were also evaluated. IGF-1 levels were elevated (P < 0.05) to a similar extent in men and women after 3 h of exercise but remained elevated at 4 h in only women. IL-6 levels were ~4-fold greater after exercise in both men and women (P < 0.05). MMP-2 levels increased to a similar extent (~2-3-fold) in men and women (P < 0.05). In contrast, MMP-9 increased with exercise but only in men (P < 0.05). Last, TIMP-1 levels also increased (P < 0.05) with exercise in men and women but the increase was more prolonged in women. In conclusion, we observed modest sex differences in tendon release of MMP-9, TIMP-1, and IGF-1 after acute resistance exercise. If such differences persist throughout a chronic exercise training, they may contribute to the reduced ability of women to adapt to exercise compared with men.NEW & NOTEWORTHY In this investigation we utilized microdialysis of the peritendinous Achilles to evaluate potential differences between men and women in tendon production of key regulators of extracellular matrix remodeling. We demonstrate that a modest sex-specific difference exists in peritendinous levels of several key extracellular matrix modulators after an acute bout of resistance exercise.

Abstract P137: Mir-122 is Associated With Stress-response Protein, Hemeoxygenase-1, for Regulation of Extracellular Matrix Remodeling in Renal Hypertension

Oxidative stress is a major contributing factor in hypertension-induced kidney injury. Hemeoxygenase-1 (Ho-1) is stress response protein constitutively expressed by the proximal tubular epithelial cells in response to oxidative stress. MicroRNAs are single stranded RNA involved in the regulation of gene expression. MicroRNA-122 has been shown to regulate Ho-1 expression in hepatitis; however whether miR-122 regulates Ho-1 in hypertensive kidney is not known. The purpose of the study was to investigate the miRNA-122 Ho-1 regulation and determine its role in extracellular matrix remodeling in renal hypertension. In vitro experiments were done using mesangial cells, treated with/without 200 μM of Angiotensin-II (Ang-II). Ho-1 was induced by ~3.5 folds with Ang-II treatment. miR-122, Ho-1 regulator, was downregulated by &gt;15 times in Ang-II treated cells. In vivo experiments were performed on WT (C57BL6/J) mice aged 12-14 wk and 75-78 wk. The animals were treated with Ang-II (1000ng/kg/min) for 4 weeks. Ho-1 is ~6 folds less in kidney of aged mice as compared to that in the young mice. Hypertension increases miR-122 expression to a greater extent (~5 folds) in aged animals. In Ho-1 knocked down mesangial cells, the extracellular matrix component, Collagen 1A1 (Col1a1), was increased by ~2 folds. In contrast, vascular endothelial growth factor ( Vegf ) and hypoxia-inducible factor ( Hif1 α ) were downregulated in Ho-1 depleted cells. In conclusion, micro RNA, miR-122, transcriptionally regulates Ho-1 as a repressor in kidney and thus affects Ho-1 mediated regulation of the extracellular remodeling in hypertension-induced renal damage.

Transforming growth factor-β1 increases lysyl oxidase expression by downregulating MIR29A in human granulosa lutein cells.

Lysyl oxidase (LOX), a key enzyme in the formation and stabilization of the extracellular matrix, is expressed in granulosa cells and plays a critical role in the regulation of granulosa cell differentiation, oocyte maturation and ovulation. To date, the regulation of LOX expression in human granulosa cells remains largely unknown. In this study, using primary and immortalized human granulosa lutein cells, we demonstrated that transforming growth factor (TGF)-β1 (TGFB1) upregulated LOX expression and downregulated microRNA-29a (MIR29A) expression via a TGF-β type I receptor-mediated signaling pathway. Additionally, we showed that MIR29A downregulated the expression of LOX in both types of cells. Furthermore, the downregulation of MIR29A contributed to the TGFB1-induced increase in LOX expression because the inhibition of MIR29A with a MIR29A inhibitor not only reversed the MIR29A-induced downregulation of LOX but also enhanced the TGFB1-induced upregulation of LOX. Our findings suggest that TGFB1 and MIR29A may play essential roles in the regulation of extracellular matrix remodeling during the periovulatory phase.

Adipose-derived stem cells increase angiogenesis through matrix metalloproteinase-dependent collagen remodeling.

Adipose-derived stem cells (ASCs) are key regulators of new blood vessel formation and widely investigated for their role in tissue regeneration and tumorigenesis. However, the cellular and molecular mechanisms through which ASCs regulate angiogenesis are not well understood. Here, it was our goal to test the functional contribution of ASC-mediated extracellular matrix (ECM) remodeling on endothelial cell invasion. To isolate the effect of ECM-remodeling, ASCs were embedded within 3-D collagen type I hydrogels and pre-cultured for 7 days; controls were not pre-cultured. A confluent monolayer of human umbilical vein endothelial cells (HUVECs) was seeded on top and its invasion into the underlying hydrogel was analyzed. Without pre-culture, ASCs inhibited vascular sprouting by stabilizing the endothelium. In contrast, 7 day pre-culture of ASCs drastically increased invasion by HUVECs. This effect was largely mediated by proteolytic ECM degradation by ASC-derived matrix metalloproteinases (MMPs) rather than vascular endothelial growth factor (VEGF), as our results indicated that blockade of MMPs, but not VEGF, inhibited endothelial sprouting. Collectively, these data suggest that the angiogenic capability of ASCs is modulated by their proteolytic remodeling of the ECM, opening new avenues for pro- and anti-angiogenic therapies.

Vascular Endothelial Growth Factor Expression in CD138+/CD19- and CD138+/CD19+ Plasma Cells in Monoclonal Gammopathies and Impact on Multiple Myeloma Prognosis

INTRODUCTIONVascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of extracellular matrix remodeling and inflammatory cytokine generation with an important role in the bone marrow microenvironment of multiple myeloma (MM). Angiogenesis is enhanced in the bone marrow of MM patients in parallel with tumor progression. Myeloma and stromal cells secrete angiogenic factors that include VEGF. Previous studies showed heterogeneity in the expression of VEGF between plasma cells (PCs) from the same MM patient. However, no clear association with expression levels, phenotypic subtypes of PCs and prognosis was demonstrated. MATHERIALS AND METHODSBone marrow PCs from 128 patients with monoclonal gammopathies, 60 patients with newly diagnosed symptomatic MM and 68 with monoclonal gammopathy of uncertain significance (MGUS) and also from 11 non-neoplastic controls (Ctr) were analysed between April 2010 and July 2013. We evaluated the expression of cytoplasmic VEGF with monoclonal antibodies by flow cytometry in the two populations of PCs, identified by gating CD138+/CD19- (clonal PCs) and CD138+/CD19+ (non-clonal PCs). The results are presented as percentage of PCs expressing VEGF and as expression levels of VEGF in mean intensity of fluorescence (MIF). The effects of VEGF expression on progression-free survival (PFS) and overall survival (OS) were analysed. For statistical analysis, software IBM SPSS Statistics v22 was used. Survival was estimated according to the Kaplan-Meier method. RESULTSIn our cohort of patients, median age was 70 (39-86) years, 52% were male. We found increased expression levels of VEGF in CD138+/CD19- PCs from MM (80 ± 7,5 MIF) compared to MGUS patients (61,7 ± 6,2 MIF) (p=0,011), as well as superior to CD138+/CD19+ PCs expression (39,92 ± 1,74 MIF) in both populations of patients (p<0,001 and p=0,02, respectively). No diferences were observed in the expression levels of VEGF in CD138+/CD19+ PCs from MM (39,92 ± 1,74 MIF), MGUS patients (41,18 ± 1,92 MIF) and controls (32,8±1,5 MIF). However, the percentage of CD138+/CD19+ expressing VEGF was significantly higher in MGUS (39,4±4%) and in MM patients (46,7±4,5%) compared to Ctr (13,5±0,5%)(p=0,019 and p=0,003, respectively). In MM patients, we also found an association between increased VEGF expression levels in CD138+/CD19- PCs (superior or equal to 175 MIF) and inferior PFS (p=0,002) and OS (p=0,003), irrespective of first line therapy (bortezomib-based regimens for fit patients or alkylating-based treatments for unfit patients). Interestingly, we also observed an incresed percentage of CD138+/CD19+ PCs (higher or equal to 21%) expressing VEGF in MM patients with a more favorable PFS (p= 0,04) and OS (p=0,008). CONCLUSIONSThe results of our investigation showed that CD138+/CD19- and CD138+/CD19+ PCs have diferences in what concerns VEGF expression, not only in MM patients, but also in MGUS patients. The increased expression of VEGF in clonal PCs from MM compared to MGUS patients evidences the relevance of VEGF in myelomagenesis. We also demonstrated a negative prognostic impact of an increased VEGF expression in CD138+/CD19- PCs, highlighting the role of VEGF in the survival and maintenance of clonal PCs and as a predictor of outcome in MM progression. The association between the percentage of CD138+/CD19+ PCs and survival supports the suggestion that these cells may not be neutral players in the complex pathogenesis of MM. The results of our study should be further investigated in larger series of patients. DisclosuresGeraldes:Celgene: Employment, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A Hypothesis for the Role of RECK in Angiogenesis.

Angiogenesis is a key process by which new capillary blood vessels are formed, sustaining the supply of oxygen and other nutrients to the body allowing its growth and wound healing, among others. However, angiogenesis also associates with pathological processes, such us tumor growth. Vascular endothelial cells produce different matrix remodeling enzymes such as matrix metalloproteinases and a-disintegrin and metalloproteinases, which have both positive and negative effects on angiogenesis, regulating the cell environment and signaling. However, little is known about the regulation of the activity of these proteases during vascular development. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored inhibitor of different matrix metalloproteinases and a-disintegrin and metalloproteinases, being a critical regulator of extracellular matrix remodeling and signaling pathway, particularly Notch, which is critical for the maturation of the growing vessels. Reck knockout mice die in utero showing vascular developmental defects and massive hemorrhages. These defects were not observed in knockout mice for secreted-soluble matrix metalloproteinase inhibitors pointing to an exclusive role of RECK in vascular development and maturation since its location at the plasma membrane. Despite the above, the exact role of RECK in this process has not been clarified. This review is focused to summarize the available information on the role of RECK as membrane anchored matrix metalloproteinases and a-disintegrin and metalloproteinases inhibitor, proposing a hypothesis by which RECK play key roles in the physiology and pathophysiology of the angiogenesis processes.

Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

P70 S6-kinase mediates the cooperation between Akt1 and Mek1 pathways in fibroblast-mediated extracellular matrix remodeling

Previous studies have demonstrated both synergistic and opposing effects of Akt and Mek1/2 in various cell functions and disease states. Furthermore, Akt has been reported to inhibit and activate cRaf/Mek pathway, suggesting that their mutual interaction and cooperation may be cell type, stimuli and/or context specific. While PI3-kinase/Akt and cRaf/Mek pathways have been implicated in the regulation of extracellular matrix (ECM) remodeling, mutual interactions between these two pathways and their specific contributions to the events leading to ECM synthesis and assembly is not clear. We investigated the specific role of Akt1 and Mek1 in ECM synthesis and assembly by NIH 3T3 fibroblasts and how these effects were reconciled to mediate overall ECM remodeling. Our study identified that cooperation between Akt1 and Mek1 is necessary to mediate ECM synthesis. Whereas Akt1 activation resulted in Mek1 activation as evidenced by increased ERK1/2 phosphorylation, Mek1 inhibition using U0126 or DN-Mek1 resulted in enhanced Akt1 phosphorylation. Interestingly, both Akt1 and Mek1 activities were needed for the synthesis and assembly of ECM. The effect of Akt1 and Mek1 on ECM synthesis was reconciled through the activation of p70 S6-kinase via phosphorylation at T421/S424 and S411, respectively. Furthermore, Akt1 and Mek1 cooperated in mediating ECM assembly via activation of integrin β1. Together, we show for the first time that Akt1 and Mek1 pathways cooperate in the regulation of ECM remodeling by the fibroblasts.

Plasma levels of matrix metalloproteinase-2, -3, -10, and tissue inhibitor of metalloproteinase-1 are associated with vascular complications in patients with type 1 diabetes: the EURODIAB Prospective Complications Study

BackgroundImpaired regulation of extracellular matrix remodeling by matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) may contribute to vascular complications in patients with type 1 diabetes. We investigated associations between plasma MMP-1, −2, −3, −9, −10 and TIMP-1, and cardiovascular disease (CVD) or microvascular complications in type 1 diabetic patients. We also evaluated to which extent these associations could be explained by low-grade inflammation (LGI) or endothelial dysfunction (ED).Methods493 type 1 diabetes patients (39.5 ± 9.9 years old, 51% men) from the EURODIAB Prospective Complications Study were included. Linear regression analysis was applied to investigate differences in plasma levels of MMP-1, −2, −3, −9, −10, and TIMP-1 between patients with and without CVD, albuminuria or retinopathy. All analyses were adjusted for age, sex, duration of diabetes, Hba1c and additionally for other cardiovascular risk factors including LGI and ED.ResultsPatients with CVD (n = 118) showed significantly higher levels of TIMP-1 [β = 0.32 SD (95%CI: 0.12; 0.52)], but not of MMPs, than patients without CVD (n = 375). Higher plasma levels of MMP-2, MMP-3, MMP-10 and TIMP-1 were associated with higher levels of albuminuria (p-trends were 0.028, 0.004, 0.005 and 0.001, respectively). Severity of retinopathy was significantly associated with higher levels of MMP-2 (p-trend = 0.017). These associations remained significant after further adjustment for markers of LGI and ED.ConclusionsThese data support the hypothesis that impaired regulation of matrix remodeling by actions of MMP-2, -3 and-10 and TIMP-1 contributes to the pathogenesis of vascular complications in type 1 diabetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0195-2) contains supplementary material, which is available to authorized users.