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Immediately following a fracture, a fibrin laden hematoma is formed to prevent bleeding and infection. Subsequently, the organized removal of fibrin, via the protease plasmin, is essential to permit fracture repair through angiogenesis and ossification. Yet, when plasmin activity is lost, the depletion of fibrin alone is insufficient to fully restore fracture repair, suggesting the existence of additional plasmin targets important for fracture repair. Previously, activated matrix metalloproteinase 9 (MMP-9) was demonstrated to function in fracture repair by promoting angiogenesis. Given that MMP-9 is a defined plasmin target, it was hypothesized that pro-MMP-9, following plasmin activation, promotes fracture repair. This hypothesis was tested in a fixed murine femur fracture model with serial assessment of fracture healing. Contrary to previous findings, a complete loss of MMP-9 failed to affect fracture healing and union through 28 days post injury. Therefore, these results demonstrated that MMP-9 is dispensable for timely fracture union and cartilage transition to bone in fixed femur fractures. Pro-MMP-9 is therefore not a significant target of plasmin in fracture repair and future studies assessing additional plasmin targets associated with angiogenesis are warranted.
OBJECT Matrix metalloprotease-9 (MMP-9) plays a critical role in infarct progression, blood-brain barrier (BBB) disruption, and vasogenic edema. While systemic administration of MMP-9 inhibitors has shown neuroprotective promise in ischemic stroke, there has been little effort to incorporate these drugs into endovascular modalities. By modifying the rodent middle cerebral artery occlusion (MCAO) model to allow local intraarterial delivery of drugs, one has the ability to mimic endovascular delivery of therapeutics. Using this model, the authors sought to maximize the protective potential of MMP-9 inhibition by intraarterial administration of an MMP-9 inhibitor, norcantharidin (NCTD). METHODS Spontaneously hypertensive rats were subjected to 90-minute MCAO followed immediately by local intraarterial administration of NCTD. The rats' neurobehavioral performances were scored according to the ladder rung walking test results and the Garcia neurological test for as long as 7 days after stroke. MRI was also conducted 24 hours after the stroke to assess infarct volume and BBB disruption. At the end of the experimental protocol, rat brains were used for active MMP-9 immunohistochemical analysis to assess the degree of MMP-9 inhibition. RESULTS NCTD-treated rats showed significantly better neurobehavioral scores for all days tested. MR images also depicted significantly decreased infarct volumes and BBB disruption 24 hours after stroke. Inhibition of MMP-9 expression in the ischemic region was depicted on immunohistochemical analysis, wherein treated rats showed decreased active MMP-9 staining compared with controls. CONCLUSIONS Intraarterial NCTD significantly improved outcome when administered at the time of reperfusion in a spontaneously hypertensive rat stroke model. This study suggests that supplementing endovascular revascularization with local neuroprotective drug therapy may be a viable therapeutic strategy.
Peripheral artery disease (PAD) is characterized by vessel occlusion and ischemia in the limbs. Treatment for PAD with surgical interventions has been showing limited success. Moreover, recent clinical trials with treatment of angiogenic growth factors proved ineffective as increased angiogenesis triggered severe inflammation in a proportionally coupled fashion. Hence, the overarching goal of this research was to address this issue by developing a biomaterial system that enables controlled, dual delivery of pro-angiogenic C16 and anti-inflammatory Ac-SDKP peptides in a minimally-invasive way. To achieve the goal, a peptide-loaded injectable microgel system was developed and tested in a mouse model of PAD. When delivered through multiple, low volume injections, the combination of C16 and Ac-SDKP peptides promoted angiogenesis, muscle regeneration, and perfusion recovery, while minimizing detrimental inflammation. Additionally, this peptide combination regulated inflammatory TNF-α pathways independently of MMP-9 mediated pathways of angiogenesis in vitro, suggesting a potential mechanism by which angiogenic and inflammatory responses can be uncoupled in the context of PAD. This study demonstrates a translatable potential of the dual peptide-loaded injectable microgel system for PAD treatment.
Copyright © 2014 Elsevier Ltd. All rights reserved.
AIMS/HYPOTHESIS - Increased extracellular matrix (ECM) collagen is a characteristic of muscle insulin resistance. Matrix metalloproteinase (MMP) 9 is a primary enzyme that degrades collagen IV (ColIV). As a component of the basement membrane, ColIV plays a key role in ECM remodelling. We tested the hypotheses that genetic deletion of MMP9 in mice increases muscle ColIV, induces insulin resistance in lean mice and worsens diet-induced muscle insulin resistance.
METHODS - Wild-type (Mmp9(+/+)) and Mmp9-null (Mmp9(-/-)) mice were chow or high-fat (HF) fed for 16 weeks. Insulin action was measured by the hyperinsulinaemic-euglycaemic clamp in conscious weight-matched surgically catheterised mice.
RESULTS - Mmp9(-/-) and HF feeding independently increased muscle ColIV. ColIV in HF-fed Mmp9(-/-) mice was further increased. Mmp9(-/-) did not affect fasting insulin or glucose in chow- or HF-fed mice. The glucose infusion rate (GIR), endogenous glucose appearance (EndoRa) and glucose disappearance (Rd) rates, and a muscle glucose metabolic index (Rg), were the same in chow-fed Mmp9(+/+) and Mmp9(-/-) mice. In contrast, HF-fed Mmp9(-/-) mice had decreased GIR, insulin-stimulated increase in Rd and muscle Rg. Insulin-stimulated suppression of EndoRa, however, remained the same in HF-fed Mmp9(-/-) and Mmp9(+/+) mice. Decreased muscle Rg in HF-fed Mmp9(-/-) was associated with decreased muscle capillaries.
CONCLUSIONS/INTERPRETATION - Despite increased muscle ColIV, genetic deletion of MMP9 does not induce insulin resistance in lean mice. In contrast, this deletion results in a more profound state of insulin resistance, specifically in the skeletal muscle of HF-fed mice. These results highlight the importance of ECM remodelling in determining muscle insulin resistance in the presence of HF diet.
Osteoarthritis (OA) of the joint is a prevalent disease accompanied by chronic, debilitating pain. Recent clinical evidence has demonstrated that central sensitization contributes to OA pain. An improved understanding of how OA joint pathology impacts upon the central processing of pain is crucial for the identification of novel analgesic targets/new therapeutic strategies. Inhibitory cannabinoid 2 (CB2) receptors attenuate peripheral immune cell function and modulate central neuro-immune responses in models of neurodegeneration. Systemic administration of the CB2 receptor agonist JWH133 attenuated OA-induced pain behaviour, and the changes in circulating pro- and anti-inflammatory cytokines exhibited in this model. Electrophysiological studies revealed that spinal administration of JWH133 inhibited noxious-evoked responses of spinal neurones in the model of OA pain, but not in control rats, indicating a novel spinal role of this target. We further demonstrate dynamic changes in spinal CB2 receptor mRNA and protein expression in an OA pain model. The expression of CB2 receptor protein by both neurones and microglia in the spinal cord was significantly increased in the model of OA. Hallmarks of central sensitization, significant spinal astrogliosis and increases in activity of metalloproteases MMP-2 and MMP-9 in the spinal cord were evident in the model of OA pain. Systemic administration of JWH133 attenuated these markers of central sensitization, providing a neurobiological basis for analgesic effects of the CB2 receptor in this model of OA pain. Analysis of human spinal cord revealed a negative correlation between spinal cord CB2 receptor mRNA and macroscopic knee chondropathy. These data provide new clinically relevant evidence that joint damage and spinal CB2 receptor expression are correlated combined with converging pre-clinical evidence that activation of CB2 receptors inhibits central sensitization and its contribution to the manifestation of chronic OA pain. These findings suggest that targeting CB2 receptors may have therapeutic potential for treating OA pain.
Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.
Duchenne muscular dystrophy (DMD) caused by loss of cytoskeletal protein dystrophin is a devastating disorder of skeletal muscle. Primary deficiency of dystrophin leads to several secondary pathological changes including fiber degeneration and regeneration, extracellular matrix breakdown, inflammation, and fibrosis. Matrix metalloproteinases (MMPs) are a group of extracellular proteases that are involved in tissue remodeling, inflammation, and development of interstitial fibrosis in many disease states. We have recently reported that the inhibition of MMP-9 improves myopathy and augments myofiber regeneration in mdx mice (a mouse model of DMD). However, the mechanisms by which MMP-9 regulates disease progression in mdx mice remain less understood. In this report, we demonstrate that the inhibition of MMP-9 augments the proliferation of satellite cells in dystrophic muscle. MMP-9 inhibition also causes significant reduction in percentage of M1 macrophages with concomitant increase in the proportion of promyogenic M2 macrophages in mdx mice. Moreover, inhibition of MMP-9 increases the expression of Notch ligands and receptors, and Notch target genes in skeletal muscle of mdx mice. Furthermore, our results show that while MMP-9 inhibition augments the expression of components of canonical Wnt signaling, it reduces the expression of genes whose products are involved in activation of non-canonical Wnt signaling in mdx mice. Finally, the inhibition of MMP-9 was found to dramatically improve the engraftment of transplanted myoblasts in skeletal muscle of mdx mice. Collectively, our study suggests that the inhibition of MMP-9 is a promising approach to stimulate myofiber regeneration and improving engraftment of muscle progenitor cells in dystrophic muscle.
OBJECTIVE - Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.
METHODS AND RESULTS - Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ-/-) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ-/- mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ-/- mice normalized flow-mediated remodeling.
CONCLUSION - CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.
STUDY QUESTION - Can biologically active vitamin D3 [1,25(OH)₂D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells?
SUMMARY ANSWER - 1,25(OH)₂D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells.
WHAT IS KNOWN ALREADY - Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)₂D3 can regulate the expression and activities of MMPs in human uterine fibroid cells.
STUDY DESIGN, SIZE, DURATION - Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells.
PARTICIPANTS/MATERIALS, SETTING, METHODS - An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)₂D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay.
MAIN RESULTS AND THE ROLE OF CHANCE - 1-1000 nM 1,25(OH)₂D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)₂D3. 1,25(OH)₂D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)₂D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)₂D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)₂D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable.
LIMITATIONS, REASONS FOR CAUTION - This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored.
WIDER IMPLICATIONS OF THE FINDINGS - This study reveals an important biological function of 1,25(OH)₂D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)₂D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.
Chemotherapeutics such as doxorubicin (DOX) and paclitaxel (PXL) have dose-limiting systemic toxicities, including cardiotoxicity and peripheral neuropathy. Delivery strategies to minimize these undesirable effects are needed and could improve efficacy, while reducing patient morbidity. Here, DOX and PXL were conjugated to a nanodendron (ND) through an MMP9-cleavable peptide linker, producing two new therapies, ND2(DOX) and ND2(PXL), designed to improve delivery specificity to the tumor microenvironment and reduce systemic toxicity. Comparative cytotoxicity assays were performed between intact ND-drug conjugates and the MMP9 released drug in cell lines with and without MMP9 expression. While ND2(DOX) was found to lose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2(PXL) was determined to have the desired properties for a prodrug delivery system. ND2(PXL) was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221A-luc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 h. Treating ND2(PXL) with MMP9 prior to the cytotoxicity assay resulted in a faster response; however, both cleaved and intact versions of the drug reached the same efficacy as the unmodified drug by 96 h in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLC(MMP9)) or do not (LLC(RSV)) express MMP9 demonstrate the selectivity of ND2(PXL) for MMP9. LLC(MMP9) cells were only 20% viable after 48 h of treatment, while LLC(RSV) were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLC(MMP9) cells with ND2(PXL) eliminated the response of the MMP9 expressing cells (LLC(MMP9)). The data presented here suggests that these NDs, specifically ND2(PXL), are nontoxic until activated by MMP9, a protease common in the microenvironment of tumors, indicating that incorporation of chemotherapeutic or cytostatic agents onto the ND platform have potential for tumor-targeted efficacy with reduced in vivo systemic toxicities.