Release Of Metalloproteinases Research Articles

Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I–IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I–III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

Cigarette Smoke and Nicotine-Induced Remodeling of Actin Cytoskeleton and Extracellular Matrix by Vascular Smooth Muscle Cells

Cigarette smoking is a significant risk factor for atherosclerosis, which involves the invasion of vascular smooth muscle cells (VSMCs) from the media to intima. Many invasive cells remodel the actin cytoskeleton to form podosomes, which regulate metalloproteinase (MMP) release for extracellular matrix (ECM) degradation. We tested the hypothesis that cigarette smoke extract (CSE) modulates the structure and function of podosomes in VSMCs. We found that, in response to PKC activation by phorbol dibutyrate (PDBu), untreated A7r5 VSMCs formed podosomes, whereas CSE, nicotine, and carbachol-treated cells formed actin-rich rings.

Cervical Remodeling/Ripening at Term and Preterm Delivery: The Same Mechanism Initiated by Different Mediators and Different Effector Cells

BackgroundPremature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice.Methodology/Principal FindingsHere we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a.Conclusion and SignificanceIn conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity.

Anti‐inflammatory mechanisms of mapracorat, a novel selective glucocorticoid receptor agonist, in human conjunctival fibroblasts

AbstractPurpose Mapracorat (BOL‐303242‐X; ZK 245186) is a selective glucocorticoid receptor agonist (SEGRA), under clinical evaluation for the treatment of inflammatory eye and skin diseases. It is structurally distinct from glucocorticoids and NSAIDs. Data suggest an improved side effect profile of this compound compared to traditional glucocorticoids. This study was to determine its anti‐inflammatory mechanisms.Methods Primary human conjunctival fibroblasts (HConF)were challenged with IL‐1ß and Luminex technology or ELISA was used to determine the effect of mapracorat on IL‐1ß‐induced cytokine, prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP) release in the presence or absence of mifepristone (RU‐486), a glucocorticoid receptor antagonist. The effect of mapracorat on IL‐1ß‐induced cyclooxygenase‐2 (COX‐2) expression was assessed by Western blotting. Dexamethasone was used as the control.Results IL‐1ß induced release of multiple cytokines, including IL‐6, IL‐8 and monocyte chemotactic protein‐1 (MCP‐1), and of PGE2 and MMPs (MMP‐1 and MMP‐3), as well as the expression of COX‐2. Mapracorat inhibited cytokine, PGE2 and MMP release as well as COX‐2 expression in a dose‐dependent manner with comparable effectiveness as dexamethasone. The inhibition of cytokine and PGE2 release was fully or partially reversed by mifepristone.Conclusion Mapracorat acts as a potent anti‐inflammatory agent in HConF by inhibiting multiple intracellular mediators. The fact that mapracorat is effective on inhibiting PGE2 pathway suggests that it may reduce post‐surgery pain. Clinical significance of these findings needs further investigation. Commercial interest

Using 18F-FDG microPET imaging to measure the inhibitory effects of Clematis chinensis Osbeck on the pro-inflammatory and degradative mediators associated with inflammatory arthritis

This study examined the modulating effects of Clematis chinensis Osbeck (Ranunculaeae) on pro-inflammatory and degradative mediators associated with inflammatory arthritis. Primary human chondrocytes (PHC) were stimulated with IL-1β or lipopolysaccharide (LPS) to induce the enhanced release of prostaglandin E(2) (PGE(2)), metalloproteinase (MMP-3 and -13), and cyclooxygenase-2 (COX-2) protein expression. The (18)F-FDG microPET imaging system was used to evaluate the anti-arthritic effects of Clematis chinensisin vivo. The acetone extracted Clematis chinensis (CC6) contained the most total saponins compared to other solvent's extracts and showed significant and dose-dependent inhibitory effects on PGE(2), MMP-3, -13, and COX-2 productions by LPS-stimulated PHC. Furthermore, CC6 also exerted inhibitory effects on 2-(18)F-fluoro-2-deoxy-d-glucose ((18)F-FDG) uptake when assessed by positron emission tomography (PET) uptake in the joints and serum PGE(2) of rabbits with knee joints injected with LPS. The results suggest the significant chondroprotective effects of Clematis chinensis are through its anti-inflammatory and MMPs inhibitory abilities. Meanwhile, we established a new analysis method to evaluate the Chinese herbal anti-arthritic effects.

Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability

Discoidin domain receptors (DDR1 and DDR2) are tyrosine kinase receptors for fibrillar collagen implicated in postnatal development, tissue repair, and primary and metastatic cancer progression. While DDR1 has been described in tumor cells, DDR2 has been localized in the tumor stroma, but its presence in the tumor cells remains unknown. The aim of this study was to elucidate the role of DDR2 signaling in tumor cells during hepatic metastasis progression. DDR2 expression and phosphorylation in cultured human A375 melanoma cells was documented by Western blot analysis. A375 cells were stably transfected with a small interfering RNA (siRNA) against DDR2 and two clones were selected: A375R2-70 and A375R2-40, with 70 and 40% of the DDR2 protein expression respectively, compared to mock-transfected cells (A375R2-100). Development of experimental liver metastasis by intrasplenic inoculation of A375R2-70 and A37R2-40 clones was reduced by 60 and 75%, respectively, measured as tumor volume, compared to livers injected with A375R2-100 cells. Accordingly, A375R2-70 and A37R2-40 clones showed reduced in vitro gelatinase activity and JNK phosphorylation, compared to mock transfected cells, with maximal inhibition in A375R2-40. Additionally, A375 melanoma, SK-HEP hepatoma and HT-29 colon carcinoma human cell lines transiently transfected with siRNA against DDR2 also showed reduced proliferation and migration rates compared to mock-transfected ones. In conclusion, DDR2 promotes A375 melanoma metastasis to the liver and the underlying mechanism implicates regulation of metalloproteinase release, cell growth and chemotactic invasion of the host tissue.

Complement Activation Triggers Metalloproteinases Release Inducing Cervical Remodeling and Preterm Birth in Mice

Inflammation is frequently linked to preterm delivery (PTD). Here, we tested the hypothesis that complement activation plays a role in cervical remodeling and PTD. We studied two mouse models of inflammation-induced PTD. The first model was induced by vaginal administration of lipopolysaccharide (LPS) and the second one by administration of progesterone antagonist RU486. Increased cervical C3 deposition and macrophages infiltration and increased serum C3adesArg and C5adesArg levels were observed in both models when compared to gestational age matched controls. A significant increase in collagen degradation, matrix metalloproteinase 9 (MMP-9) activity and tissue distensibility was observed in the cervix in both models. Mice deficient in complement receptor C5a did not show increased MMP-9 activity and cervical remodeling and did not deliver preterm in response to LPS or RU486, suggesting a role for C5aR in the cervical changes that precede PTD. In vitro studies show that macrophages release MMP-9 in response to C5a. Progesterone diminished the amount of C5aR on the macrophages surface, inhibited the release of MMP-9 and prevented PTD. In addition, macrophages depletion also prevented cervical remodeling and PTD in LPS-treated mice. Our studies show that C5a-C5aR interaction is required for MMP-9 release from macrophages, and the cervical remodeling that leads to PTD. Complement inhibition and supplementation with progesterone may be good therapeutic options to prevent this serious pregnancy complication.

A2A and A3 adenosine receptor expression in rheumatoid arthritis: upregulation, inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release.

IntroductionThe reduction of the inflammatory status represents one of the most important targets in rheumatoid arthritis (RA). A central role of A2A and A3 adenosine receptors (ARs) in mechanisms of inflammation has been reported in different pathologies. The primary aim of this study was to investigate the A2A and A3ARs and their involvement in RA progression measured by Disease Activity Score in 28 or 44 joints (DAS28 or DAS).MethodsARs were analyzed by saturation binding assays, mRNA and Western blotting analysis in lymphocytes from early and established RA patients. The effect of A2A and A3AR agonists in nuclear factor kB (NF-kB) pathway was evaluated. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) release was carried out by A2A and A3AR activation. AR pharmacological regulation in matrix metalloproteinase-1 (MMP-1) and metalloproteinase-3 (MMP-3) release was also studied.ResultsIn lymphocytes obtained from RA patients, A2A and A3ARs were up-regulated if compared with healthy controls. A2A and A3AR activation inhibited the NF-kB pathway and diminished inflammatory cytokines such as TNF-α, IL-1β and IL-6. A2A and A3AR agonists mediated a reduction of MMP-1 and MMP-3 release. A2A and A3AR density inversely correlated with DAS28 and DAS suggesting a direct role of the endogenous activation of these receptors in the control of RA joint inflammation.ConclusionsTaken together these data demonstrate that the inflammatory and clinical responses in RA are regulated by A2A and A3ARs and support the use of A2A and/or A3AR agonists as novel and effective pharmacological treatment in RA patients.

Simvastatin as a Modulator of Tissue Remodeling through Inhibition of Matrix Metalloproteinase (MMP) Release from Human Lung Fibroblasts

Methods: This study, using an in-vitro model (three-dimensional collagen gel contraction system), evaluated the effect of cytokines (tumor necrosis factor-α, TNF-a and interleukin-1β, IL-1b) on the MMP release and MMP activation from human lung fibroblasts. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Results: In three-dimensional collagen gel cultures (3D cultures) where cytokines (TNF-a and IL-1b) can induce the production of MMPs by fibroblasts, it was found that simvastatin inhibited MMP release. In 3D cultures, cytokines together with NE induced collagen degradation and can lead to activation of the MMP, which was inhibited by simvastatin. Conclusion: Simvastatin may play a role in regulating human lung fibroblast functions in repair and remodeling processes by inhibiting MMP release and the conversion from the latent to the active form of MMP.

Blood-born tissue factor in cardiovascular disease: where are we now?

Over the last decade, many clinical, pathological and laboratory data have been obtained indicating that, in the majority of cases, acute coronary thrombosis occurs upon rupture of the thin fibrous cap of a vulnerable atherosclerotic plaques, a result of a series of immuno-inflammatory processes. The inner content of the plaque becomes then exposed to the circulating blood leading to the deposition of thrombotic material into the lumen [1]. Our data, as well as that acquired by others, demonstrate that one of the major determinants of the thrombogenicity of the disrupted atheroma is tissue factor (TF),which triggers the activation of coagulation by binding to factor VII/VIIa. TF is particularly abundant in the lipid-rich necrotic core of the lesion, which is therefore the most thrombogenic component of atherosclerotic plaque [2]. The binding of TF to factor VIIa causes thrombin generation, which in turn induces platelet activation and fibrin formation leading to a partial or total thrombotic occlusion of the artery, and to the occurrence of an acute coronary syndrome (ACS) [1]. We also have demonstrated that the activation of the coagulation cascade within the coronary vessel at sites of atherosclerotic plaque rupture is largely TF-dependent, since a significant reduction in both platelet and fibrin deposition can be obtained by local pretreatment of atherosclerotic lesions with the recombinant analog of the physiologic tissue factor pathway inhibitor (rTFPI) or with a specific anti-TF antibody [3]. Instabilization and rupture of the plaque are mainly due to inflammatory processes that take place in the vessel wall. Experimental studies demonstrate that inflammatory cells such as macrophages, macrophage-derived lipid laden ‘foam cells’, and T lymphocytes are major constituents of plaques, and infiltrate areas of superficial erosion and the shoulder region of the coronary lesions where the fibrous cap usually breaks [4]. Macrophages and ‘foam cells’ are also the major source of TF within the lesions, and therefore this protein can be considered a product of the inflammatory processes of the atheroma [1]. The interplay between these cells results in the production and local secretion of several inflammatory cytokines and chemokines like interleukin (IL)-1, interferon-c and TNF-a, which, in turn, lead to the release of metalloproteinases (MMPs), particularly of MMP-9, which degrade extracellular matrix and particularly collagen, thus inducing plaque weakening and fracture[1, 5, 6].

Circulating matrix metalloproteinase levels after ventricular septal defect repair in infants

Surgery for congenital heart disease initiates a complex inflammatory response that can influence the postoperative course. However, broad integration of the cytokine and proteolytic cascades (matrix metalloproteinases: MMPs), which may contribute to postoperative outcomes, has not been performed. Using a low-volume (50-60 μL), high-sensitivity, multiplex approach, we serially measured a panel of cytokines (interleukins 2, 4, 6, 8, and 10, tumor necrosis factor alpha, interleukin 1β, and granulocyte-macrophage colony stimulating factor) and matrix metalloproteinases (matrix metalloproteinases 2, 3, 7, 8, 9, 12, and 13) in patients (n = 9) preoperatively and after repair of ventricular septal defect. Results were correlated with outcomes such as inotropic requirement, oxygenation, and fluid balance. Serial changes in perioperative plasma levels of the cytokines and matrix metalloproteinases exhibited distinct temporal profiles. Plasma levels of interleukins 2, 8, and 10 and matrix metalloproteinase 9 peaked within 4 hours, whereas levels of matrix metalloproteinase 3 and 8 remained elevated at 24 and 48 hours after crossclamp removal. Area-under-the-curve analysis of early cytokine levels were associated with major clinical variables, including inverse correlations between early interleukin 10 levels and cumulative inotrope requirement at 48 hours (r: -0.85; P < .005) and late matrix metalloproteinase 7 levels and cumulative fluid balance (r: -0.90; P < .001). The unique findings of this study were that serial profiling a large array of cytokines and proteolytic enzymes after surgery for congenital heart disease can provide insight into relationships between changes in bioactive molecules to early postoperative outcomes. Specific patterns of cytokine and matrix metalloproteinase release may hold significance as biomarkers for predicting and managing the postoperative course after surgery for congenital heart disease.

Chondrocytes from arthritic cartilage in a subset of osteoarthritis patients express lower levels of NFκB-P65: a survival factor against tumour necrosis factor α-induced cell death

Chondrocytes play a central role in cartilage pathology, as seen in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), by a deranged synthesis of extracellular matrix (ECM) components and the...

Liver graft exposure to carbon monoxide during cold storage protects sinusoidal endothelial cells and ameliorates reperfusion injury in rats

Hepatic ischemia/reperfusion (I/R) injury significantly influences short-term and long-term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO-UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI(+) SECs was significantly lower in grafts stored in CO-UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO-UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO-UW solution improved to 80% (8/10). These beneficial effects of CO-UW solution were associated with a significant reduction of neutrophil extravasation, down-regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal-regulated kinase activation. Liver grafts from Kupffer cell-depleted donors or pseudogerm-free donors showed less SEC death during cold preservation, and CO-UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury.

Statins inhibit matrix metalloproteinase release from human lung fibroblasts

Pleiotropic effects of statins have been reported to include inhibition of matrix metalloproteinase (MMP) release from macrophages and endothelial cells. We evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodelling processes. Monolayer and three-dimensional (3D) collagen gel cultures of fibroblasts were used. Cytokines (tumour necrosis factor-alpha and interleukin-1alpha) were used to induce MMP release and mRNA expression. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Atorvastatin inhibited MMP-1 and -3 release and mRNA expression in both culture systems. Similar results were obtained with simvastatin and fluvastatin. In 3D cultures where cytokines also stimulated MMP-9 release, atorvastatin also inhibited MMP-9 release. In 3D cultures, cytokines together with NE induced collagen degradation, which was also inhibited by atorvastatin. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate. The current data suggest that statins may modulate remodelling processes mediated by fibroblasts by inhibiting MMP release.

Chondrogenic Priming of Human Bone Marrow Stromal Cells: A Better Route to Bone Repair?

The use of bioengineered cell constructs for the treatment of bone defects has received much attention of late. Often, bone marrow stromal cells (BMSCs) are used that are in vitro-stimulated toward the osteogenic lineage, aiming at intramembranous bone formation. The success of this approach has been disappointing. A major concern with these constructs is core degradation and necrosis caused by lack of vascularization. We hypothesized that stimulation of cells toward the endochondral ossification process would be more successful. In this study, we tested how in vitro priming of human BMSCs (hBMSCs) along osteogenic and chondrogenic lineages influences survival and osteogenesis in vivo. Scaffolds that were pre-cultured on chondrogenic culture medium showed collagen type II and collagen type X production. Moreover, vessel ingrowth was observed. Priming along the osteogenic lineage led to a mineralized matrix of poor quality, with few surviving cells and no vascularization. We further characterized this process in vitro using pellet cultures. In vitro, pellets cultured in chondrogenic medium showed progressive production of collagen type II and collagen type X. In the culture medium of these chondrogenic cultured pellets, vascular endothelial growth factor (VEGF) release was observed at days 14, 21, and 35. When pellets were switched to culture medium containing beta-glycerophosphate, independent of the presence or absence of transforming growth factor beta (TGF-beta), mineralization was observed with a concomitant reduction in VEGF and matrix metalloproteinase (MMP) release. By showing that VEGF and MMPs are produced in chondrogenically differentiated hBMSCs in vitro, we demonstrated that these cells produce factors that are known to be important for the induction of vascularization of the matrix. Inducing mineralization in this endochondral process does, however, severely diminish these capacities. Taken together, these data suggest that optimizing chondrogenic priming of hBMSCs may further improve vessel invasion in bioengineered constructs, thus leading to an alternative and superior approach to bone repair.

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear "impaired" wound model. To impair wound healing, macrophages were depleted by pretreatment with iota-carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor-alpha (TNF-alpha) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP-2 expression in wounds. In contrast, TNF-alpha, MMP-9, and TIMP-1 were significantly up-regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF-alpha production and, to a lesser degree due to release of metalloproteinases.

Intracellular Signaling of LOX-1 in Endothelial Cell Apoptosis

Atherosclerosis in the coronary artery is a major culprit in unstable coronary syndromes and sudden cardiac death. Rupture of the cap covering the atherosclerotic plaque is thought to lead to platelet deposition and formation of an occlusive thrombus resulting in cessation of blood flow. Oxidatively modified LDL (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and plaque rupture by promoting lipid accumulation, proinflammatory response, release of metalloproteinases, and apoptotic cell death.1,2 Ox-LDL leads to endothelial activation, characterized by expression of adhesion molecules, contributing to adherence and migration of inflammatory cells across the endothelial barrier. Endothelial activation is followed by endothelial dysfunction and injury characterized by loss of expression and activity of constitutive endothelial nitric oxide synthase and a state of oxidative stress. Ox-LDL, particularly in large concentrations, induces apoptosis in endothelial cells, macrophages, and smooth muscle cells.3–5 Ox-LDL is taken up by monocytes/macrophages and smooth muscle cells through a variety of scavenger receptors (SRs), such as SR-AI/II, CD36, SR-BI, macrosialin/CD68, SREC, and LOX-1, and exerts its proatherogenic effects on the vessel wall.1,6,7 Several studies have demonstrated that SRs other than LOX-1 are absent or expressed in minimal amounts in endothelial cells.6–8 LOX-1 plays a crucial role in ox-LDL–induced pathological transformation of the vessel wall components. The importance of LOX-1 became evident in studies that showed significant limitation of atherosclerotic lesion formation in the LDL receptor knockout mice with LOX-1 deletion.9 In these studies, there was clear evidence of preservation of endothelial function and reduction in oxidative stress and inflammatory response. LOX-1 is a type II membrane protein ox-LDL receptor with a C-type lectin-like extracellular domain and a short cytoplasmic tail.7,10 LOX-1 is minimally expressed in endothelial cells, monocytes, platelets, and smooth muscle cells under physiological conditions but highly induced under pathological conditions, such …

Mechanisms of Delayed Wound Healing by Commonly Used Antiseptics

The cytotoxic effects of antiseptics on pivotal cell types of the healing process have been well documented. The purpose of our investigation was to explore the ability of subcytotoxic levels of antiseptics to interfere with fibroblast function. Cell proliferation assays were performed by culturing fibroblasts in the presence of commonly used antiseptics. Migration was evaluated using scratch assays in which monolayers were "wounded" and cellular movement was monitored by digital photography. Matrix metalloproteinase (MMP) release was analyzed by zymography. H2O2 and povidone-iodine reduced both migration and proliferation of fibroblasts in a dose-dependent fashion. Treatment with silver-containing antiseptics and chlorhexidine exhibited reductions in proliferation at high concentrations, but enhanced growth at lower doses. Silver-containing compounds and chlorhexidine also proved to be the least detrimental to migration in these assays. metalloproteinase release from the cells was differently affected depending on the dosage and class of antiseptic applied. When debridement of the wound bed is not sufficient to reduce bacterial loads, the application of broad-spectrum antiseptics maybe indicated. Our data would suggest that H2O2 and iodine are poor choices, potentially retarding the contribution of fibroblasts to the healing process. Silver sulfadiazine and chlorhexidine, at levels still proven to be bactericidal, had fewer detrimental effects on fibroblast activity in these assays. The silver-containing antiseptics may even increase the proliferative potential of these cells in culture.

Chondrogenic Priming of Human Bone Marrow Stromal Cells: A Better Route to Bone Repair?

The use of bioengineered cell constructs for the treatment of bone defects has received much attention of late. Often, bone marrow stromal cells (BMSCs) are used that are in vitro-stimulated toward the osteogenic lineage, aiming at intramembranous bone formation. The success of this approach has been disappointing. A major concern with these constructs is core degradation and necrosis caused by lack of vascularization. We hypothesized that stimulation of cells toward the endochondral ossification process would be more successful. In this study, we tested how in vitro priming of human BMSCs (hBMSCs) along osteogenic and chondrogenic lineages influences survival and osteogenesis in vivo. Scaffolds that were pre-cultured on chondrogenic culture medium showed collagen type II and collagen type X production. Moreover, vessel ingrowth was observed. Priming along the osteogenic lineage led to a mineralized matrix of poor quality, with few surviving cells and no vascularization. We further characterized this process in vitro using pellet cultures. In vitro, pellets cultured in chondrogenic medium showed progressive production of collagen type II and collagen type X. In the culture medium of these chondrogenic cultured pellets, vascular endothelial growth factor (VEGF) release was observed at days 14, 21, and 35. When pellets were switched to culture medium containing beta-glycerophosphate, independent of the presence or absence of transforming growth factor beta (TGF-beta), mineralization was observed with a concomitant reduction in VEGF and matrix metalloproteinase (MMP) release. By showing that VEGF and MMPs are produced in chondrogenically differentiated hBMSCs in vitro, we demonstrated that these cells produce factors that are known to be important for the induction of vascularization of the matrix. Inducing mineralization in this endochondral process does, however, severely diminish these capacities. Taken together, these data suggest that optimizing chondrogenic priming of hBMSCs may further improve vessel invasion in bioengineered constructs, thus leading to an alternative and superior approach to bone repair.

Response to Letter Regarding Article, “Ablation of Matrix Metalloproteinase-9 Increases Severity of Viral Myocarditis in Mice”

It is now clear that release of metalloproteinase–9 (MMP-9)2 inhibition by tissue inhibitor of MMP-1 (TIMP-1)1 is beneficial during the acute phase of CVB3 myocarditis, but chronic MMP activation is detrimental3 after infection has been cleared. We investigated the course of coxsackievirus B3 (CVB3)–induced viral myocarditis within a murine MMP-9 knockout model.2 On accumulating initial data, it was clear that a detailed study was required to support our observation that MMP-9 was protective during acute CVB3 infection and myocarditis. We noted that CVB3 infection within the pancreas and liver was also higher in the MMP-9 …