Matrix Metalloproteinase-9 Knockout Research Articles

Matrix Metalloproteinase-9 Contributes to Epilepsy Development after Ischemic Stroke in Mice.

Epilepsy, a neurological disorder affecting over 50 million individuals globally, is characterized by an enduring predisposition and diverse consequences, both neurobiological and social. Acquired epilepsy, constituting 30% of cases, often results from brain-damaging injuries like ischemic stroke. With one third of epilepsy cases being resistant to existing drugs and without any preventive therapeutics for epileptogenesis, identifying anti-epileptogenic targets is crucial. Stroke being a leading cause of acquired epilepsy, particularly in the elderly, prompts the need for understanding post-stroke epileptogenesis. Despite the challenges in studying stroke-evoked epilepsy in rodents due to poor long-term survival rates, in this presented study the use of an animal care protocol allowed for comprehensive investigation. We highlight the role of matrix metalloproteinase-9 (MMP-9) in post-stroke epileptogenesis, emphasizing MMP-9 involvement in mouse models and its potential as a therapeutic target. Using a focal Middle Cerebral Artery occlusion model, this study demonstrates MMP-9 activation following ischemia, influencing susceptibility to seizures. MMP-9 knockout reduces epileptic features, while overexpression exacerbates them. The findings show that MMP-9 is a key player in post-stroke epileptogenesis, presenting opportunities for future therapies and expanding our understanding of acquired epilepsy.

Levofloxacin alleviates blood-brain barrier disruption following cerebral ischemia and reperfusion via directly inhibiting A-FABP

Reperfusion therapy is currently the most effective treatment for acute ischemic stroke, but often results in secondary brain injury. Adipocyte fatty acid-binding protein (A-FABP, FABP4, or aP2) was shown to critically mediate cerebral ischemia/reperfusion (I/R) injury by exacerbating blood-brain barrier (BBB) disruption. However, no A-FABP inhibitors have been approved for clinical use due to safety issues. Here, we identified the therapeutic effect of levofloxacin, a widely used antibiotic displaying A-FABP inhibitory activity in vitro, on cerebral I/R injury and determined its target specificity and action mechanism in vivo. Using molecular docking and site-directed mutagenesis, we showed that levofloxacin inhibited A-FABP activity through interacting with the amino acid residue Asp76, Gln95, Arg126 of A-FABP. Accordingly, levofloxacin significantly inhibited A-FABP-induced JNK phosphorylation and expressions of proinflammatory factors and matrix metalloproteinase 9 (MMP-9) in mouse primary macrophages. In wild-type mice with transient middle cerebral artery occlusion, levofloxacin substantially mitigated BBB disruption and neuroinflammation, leading to reduced cerebral infarction, alleviated neurological outcomes, and improved survival. Mechanistically, levofloxacin decreased MMP-9 expression and activity, and thus reduced degradation of extracellular matrix and endothelial tight junction proteins. Importantly, the BBB- and neuro-protective effects of levofloxacin were abolished in A-FABP or MMP-9 knockout mice, suggesting that the therapeutic effects of levofloxacin highly depended on specific targeting of the A-FABP-MMP-9 axis. Overall, our study demonstrates that levofloxacin alleviates A-FABP-induced BBB disruption and neural tissue injury following cerebral I/R, and unveils its therapeutic potential for the treatment of ischemic stroke.

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

Elucidating the role of MMP9 in hyperhomocysteinemia induced degradation of the neurovascular unit

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. Hyperhomocysteinemia has been identified as a risk factor for VCID though the mechanism behind this connection has yet to be determined. Hyperhomocysteinemia increases inflammation inducing the upregulation of matrix metalloproteinase 9 (MMP9) activity. MMP9 has been shown to degrade several substrates along the neurovascular unit including claudin 5, collagen 4, occludin and β‐dystroglycan. I hypothesize that increased MMP9 activity following the induction of hyperhomocysteinemia leads to the cleavage of important anchoring proteins along the neurovascular unit weakening the integrity of the blood brain barrier which contributes to the progression of VCID pathology.MethodC57BL6 WT and MMP9 knockout mice were placed on a control diet or a diet deficient in vitamins B6, B12 and folate and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12 and 16 weeks. Western blot analysis was performed to investigate the substrates of MMP9 including claudin, occludin, and β‐dystroglycan.ResultStudies are underway to examine if MMP9 activation contributes to blood brain barrier dysfunction directly by degrading tight junction proteins leading to a weakened endothelial cell barrier and/or indirectly by cleaving proteins responsible for maintaining the connection between astrocytic end feet and the endothelial basement membrane.ConclusionOur findings suggest that increased levels of MMP9 activity may impact several aspects of the neurovascular unit to contribute to VCID pathology. Further investigations focused on inhibition of MMP9 activation could provide the field with an important target for mitigating progression of this disease.

Clostridium butyricum Protects Against Pancreatic and Intestinal Injury After Severe Acute Pancreatitis via Downregulation of MMP9.

Background: Evidence have shown that gut microbiota plays an important role in the development of severe acute pancreatitis (SAP). In addition, matrix metalloproteinase-9 (MMP9) plays an important role in intestinal injury in SAP. Thus, we aimed to determine whether gut microbiota could regulate the intestinal injury during SAP via modulating MMP9. Methods: In this study, the fecal samples of patients with SAP (n = 72) and healthy controls (n = 32) were analyzed by 16S rRNA gene sequencing. In addition, to investigate the association between gut microbiota and MMP9 in intestinal injury during SAP, we established MMP9 stable knockdown Caco2 and HT29 cells in vitro and generated a MMP9 knockout (MMP9−/−) mouse model of SAP in vivo. Results: We found that the abundance of Clostridium butyricum (C. butyricum) was significantly decreased in the SAP group. In addition, overexpression of MMP9 notably downregulated the expressions of tight junction proteins and upregulated the expressions of p-p38 and p-ERK in Caco2 and HT29 cells (p < 0.05). However, C. butyricum or butyrate treatment remarkably upregulated the expressions of tight junction proteins and downregulated the expressions of MMP9, p-p38 and p-ERK in MMP9-overexpressed Caco2 and HT29 cells (p < 0.05). Importantly, C. butyricum or butyrate could not affect the expressions of tight junction proteins, and MMP9, p-p38 and p-ERK proteins in MMP9-knockdown cells compared with MMP9-knockdown group. Consistently, C. butyricum or butyrate could not attenuate pancreatic and intestinal injury during SAP in MMP9−/− mice compared with the SAP group. Conclusion: Collectively, C. butyricum could protect against pancreatic and intestinal injury after SAP via downregulation of MMP9 in vitro and in vivo.

MMP-9 Deletion Attenuates Arteriovenous Fistula Neointima through Reduced Perioperative Vascular Inflammation

Matrix metalloproteinase 9 (MMP-9) expression is upregulated in vascular inflammation and participates in vascular remodeling, including aneurysm dilatation and arterial neointima development. Neointima at the arteriovenous (AV) fistula anastomosis site primarily causes AV fistula stenosis and failure; however, the effects of MMP-9 on perioperative AV fistula remodeling remain unknown. Therefore, we created AV fistulas (end-to-side anastomosis) in wild-type (WT) and MMP-9 knockout mice with chronic kidney disease to further clarify this. Neointima progressively developed in the AV fistula venous segment of WT mice during the four-week postoperative course, and MMP-9 knockout increased the lumen area and attenuated neointima size by reducing smooth muscle cell and collagen components. Early perioperative AV fistula mRNA sequencing data revealed that inflammation-related gene sets were negatively enriched in AV fistula of MMP-9 knockout mice compared to that in WT mice. qPCR results also showed that inflammatory genes, including tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), were downregulated. In addition, Western blot results showed that MMP-9 knockout reduced CD44 and RAC-alpha serine/threonine-protein kinase (Akt) and extracellular signal-regulated kinases (ERK) phosphorylation. In vitro, MMP-9 addition enhanced IL-6 and MCP-1 expression in vascular smooth muscle cells, as well as cell migration, which was reversed by an MMP-9 inhibitor. In conclusion, MMP-9 knockout attenuated AV fistula stenosis by reducing perioperative vascular inflammation.

Silencing matrix metalloproteinase 9 exerts a protective effect on astrocytes after oxygen-glucose deprivation and is correlated with suppression of aquaporin-4

Cerebral ischemia, followed by brain edema, can be life-threatening. It has been widely reported that matrix metalloproteinase-9 (MMP-9) and aquaporin-4 (AQP4) have prominent roles in the development of brain edema. However, the exact mechanisms by which MMP-9 and AQP4 influence brain edema are not fully understood.In this study, astrocytes were subjected to oxygen-glucose deprivation (OGD) /reperfusion (OGD/R) injury, an in vitro model of Ischemia/reperfusion (I/R). Cell viability was evaluated through the measurement of LDH release. The expression of MMP-9 and AQP4 also were measured by qPCR and western blot. Subsequently, we knocked out the MMP-9 gene using MMP-9 siRNA. AQP4 and its gene expression, and the LDH release rate were measured using ELISA, Western blotting, and RT-PCR. We also assessed cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMK II) in MMP-9 knockout astrocytes. All measurements were performed with or without an OGD/R challenge.OGD/reperfusion enhanced LDH release levels, and also increased MMP-9 and AQP4 expression in astrocytes. Silencing the MMP-9 gene decreased LDH release levels, and also was associated with decreased AQP4 expression. The expression of PKC, but not PKA, PKG, or CaMK II, was decreased. This study revealed that OGD/reperfusion could cause cell damage in vitro. MMP-9 silencing protected astrocytes from hypoxic insult, and the protective effect may be enhanced by the downregulation of AQP4 expression. In conclusion, downregulating MMP-9 expression may be useful for the prevention and treatment of brain ischemia.

A pilot study of microRNA expression profiles of the spinal neuron in matrix metalloproteinase-9 knockout mice

Introduction: Matrix metalloproteinase-9 (MMP9) plays a key role in blood–spinal cord barrier dysfunction. MMP9 blockade leads to improved injured locomotor recovery. However, it is still unknown whether MMP9 deficiency affects gene expression or signal transduction pathways in the spinal neuron. Material and Methods: In this study, we first screen the MMP9 knockdown mice with high superoxide dismutase (SOD) expression and low MMP9 expression by polymerase chain reaction analysis. Then, the gene microarrays were used to screen differentially expressed genes in the spinal neuron from MMP-9 knockout and wild-type mice. There were six groups in this experiment, including three negative control groups: SOD_1, SOD_2, and SOD_3, and three experiment groups: SOD_ MMP9_1, SOD_ MMP9_2, and SOD_ MMP9_3. The gene ontology terms were used to predict the potential functions of these differentially expressed genes, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the potential functions of these target genes in the pathways. Results: We found that the gene expression in the spinal neuron from MMP9 knockout mice was significantly altered compared to wild-type mice. FoxO signaling, axon guidance, ubiquitin-mediated proteolysis, regulation of actin cytoskeleton, and proteoglycans were changed. Discussion and Conclusion: In summary, MMP9 plays a role in spinal neuron signaling and the underlying mechanism may through affecting several signaling pathways.

Effect of MMP-9 gene knockout on retinal vascular form and function.

Retinal degeneration from inherited gene mutation(s) is a common cause of blindness because of structural and functional alterations in photoreceptors. Accordingly, various approaches are being tested to ameliorate or even cure neuroretinal blinding conditions in susceptible patients by employing neuroprotective agents, gene therapeutics, optogenetics, regenerative therapies, and retinal prostheses. The FVB/NJ mouse strain inherently has a common Pde6b rd1 homozygous allele that renders its progeny blind by the time pups reach weaning age. To study the role matrix metalloproteinase-9 (MMP-9) in retinal structure and function, we examined a global MMP-9 knockout (KO) mouse model that has been engineered on the same FVB/NJ background to test the hypothesis whether lack of MMP-9 activity diminishes neuroretinal degenerative changes and thus helps improve the vision. We compared side-by-side various aspects of the ocular physiology in the wild-type (WT) C57BL/6J, FVB/NJ, and MMP-9 KO strains of mice. The results suggest that MMP-9 KO mice display subdued changes in their retinae as reflected by both structural and functional enhancement in the overall ocular neurophysiological parameters. Altogether, the findings appear to have clinical relevance for targeting conditions wherein MMPs and their overactivities are suspected to play dominant pathophysiological roles in advancing neurodegenerative retinal diseases.

Bone Marrow Niche-Derived Extracellular Matrix-Degrading Enzymes Influence the Progression of B-Cell Acute Lymphoblastic Leukemia

Interactions with the bone marrow microenvironment (BMM) influence the pathophysiology of hematological malignancies. Reciprocally, hematological malignancies remodel the BMM in order to render it more hospitable for malignant progression. Hypothesizing that different leukemias differentially remodel stroma cells in the BMM, we tested differences in the expression of various genes in sorted osteoblastic cells from mice with B-cell acute lymphoblastic (B-ALL), acute myeloid (AML) or chronic myeloid leukemia (CML) or control mice by microarray. This revealed a high expression of various proteases, especially matrix metalloproteinase-9 (MMP-9), in osteoblastic cells isolated from mice with B-ALL. MMP-9 had previously been shown to be produced by leukemia cells.To investigate the physiological relevance of BMM-derived MMP-9 for the progression of B-ALL, we employed the well-established retroviral transduction/transplantation model of BCR-ABL1-dependent B-ALL in MMP-9-deficient or wildtype mice. Deficiency of MMP-9 in the BMM prolonged the survival of mice with B-ALL compared to wildtype mice. Secondary transplantation of bone marrow from wildtype versus MMP-9 knockout (KO) mice with B-ALL led to a significant prolongation of survival of recipients of B-ALL bone marrow from MMP-9 KO mice, suggesting that leukemia-initiating cells are reduced in a MMP-9 KO BMM. In addition, we observed reduced invasion of B-ALL cells into different organs such as spleen, meninges, lungs and liver, which likely underlies the observed prolongation of survival, as well as the reduced degradation of extracellular matrix (ECM) proteins such as laminin and fibronectin in the BMM of MMP-9 deficient mice. Mechanistically, using various in vivo and in vitro assays, as well as mice deficient for the receptor for tumor necrosis factor-α (TNF-α) we have demonstrated that B-ALL cells induce MMP-9-expression in niche cells, such as mesenchymal stem cells (MSC), via a release of high amounts of TNF-α, both in the murine and the human setting. TNF-α-dependent induction of MMP-9 in MSC was mediated via TNF-receptor-1, followed by activation of the downstream target nuclear factor kappa B (NF-kB). Consistently, transplantation of B-ALL-initiating cells, in which TNF-α had been knocked down, led to a significant prolongation of survival in B-ALL mice.Testing whether treatment of mice with B-ALL with an inhibitor of MMP-9 may prolong survival compared to vehicle-treated mice, we indeed found decreased degradation of ECM proteins in the BMM, a decreased leukemia burden in peripheral blood and a significant prolongation of survival. In addition, we observed reduced minimal residual disease in the bone marrow of mice with B-ALL when the mice had been treated with cytarabine and the MMP-9 inhibitor compared to mice treated with cytarabine alone. Experiments on testing the effect of MMP-9 inhibitor treatment on human B-ALL cells in a xenotransplantation model are currently ongoing.In summary, we have demonstrated the role of BMM-derived MMP-9 for B-ALL progression. B-ALL cells self-reinforce the BMM by inducing the production of MMP-9 via the release of TNF-α. Increased production of MMP-9 from the BMM promotes B-ALL progression via increased invasion of leukemic cells into different organs. Our data suggest, that inhibition of MMP-9 may be a feasible strategy as an adjunct to existing therapies. DisclosuresKrause:Glycotope: Consultancy; Glycomimetics Inc. until 06/2016: Consultancy, Research Funding; Merck KgGA in future: Research Funding; European Patent: Patents & Royalties: European Patent No. 16187926.7-1401 “FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA” .

Elevation of MMP-9 Levels Promotes Epileptogenesis After Traumatic Brain Injury

Posttraumatic epilepsy (PTE) is a recurrent seizure disorder that often develops secondary to traumatic brain injury (TBI) that is caused by an external mechanical force. Recent evidence shows that the brain extracellular matrix plays a major role in the remodeling of neuronal connections after injury. One of the proteases that is presumably responsible for this process is matrix metalloproteinase-9 (MMP-9). The levels of MMP-9 are elevated in rodent brain tissue and human blood samples after TBI. However, no studies have described the influence of MMP-9 on the development of PTE. The present study used controlled cortical impact (CCI) as a mouse model of TBI. We examined the detailed kinetics of MMP-9 levels for 1 month after TBI and observed two peaks after injury (30 min and 6 h after injury). We tested the hypothesis that high levels of MMP-9 predispose individuals to the development of PTE, and MMP-9 inhibition would protect against PTE. We used transgenic animals with either MMP-9 knockout or MMP-9 overexpression. MMP-9 overexpression increased the number of mice that exhibited TBI-induced spontaneous seizures, and MMP-9 knockout decreased the appearance of seizures. We also evaluated changes in responsiveness to a single dose of the chemoconvulsant pentylenetetrazol. MMP-9-overexpressing mice exhibited a significantly shorter latency between pentylenetetrazol administration and the first epileptiform spike. MMP-9 knockout mice exhibited the opposite response profile. Finally, we found that the occurrence of PTE was correlated with the size of the lesion after injury. Overall, our data emphasize the contribution of MMP-9 to TBI-induced structural and physiological alterations in brain circuitry that may lead to the development of PTE.

Abstract 097: Knockout of Matrix Metalloproteinase 9 Protects Against Hypertension-induced Renal Disease in Hypertensive Dahl S Rats

Matrix metalloproteinase 9 (MMP9) is a member of gelatinase family of enzymes with potentially opposing actions on tissue fibrosis (i.e. the degradation of extracellular matrix). Our previous study showed that two nonselective MMP inhibitors, XL081 and XL784, which inhibit MMP2, 9, 13 and Adam10 could delay and even reverse the development of renal fibrosis in hypertensive Dahl Salt sensitive (SS) rats. However, the specific role of MMP9 to the development of hypertensive nephropathy in MMP9 knockout (KO) rat models is unknown. In the present study, MMP9 KO rats were created on the SS genetic background using a CRISPR/Cas 9 system and the effect of KO was verified in this model. The systolic pressure, diastolic pressure and mean blood pressure (MAP) were similar in 12 weeks old MMP9 KO (n=6) and SS rats (n=9) fed a low salt diet (145 ± 3 vs. 148 ± 2mmHg; 111 ± 2 vs. 107 ± 2mmHg; 128 ± 2 vs. 126 ± 2mmHg). MAP increased to 180 ± 4 vs. 154 ± 3mmHg in SS rats versus MMP9 KO rats fed 8% high salt (HS) diet for 3 weeks. Proteinuria increased from 196 ± 18 mg/day to 718 ± 61mg/day in HS treated SS rats (n=9). It was significantly reduced in HS treated MMP9 KO rats (211 ± 30mg/day, n=6). The degree of glomerular injury (2.88 ± 0.08 vs. 3.52 ± 0.02), interstitial fibrosis (4.57 ± 0.35% vs. 10.45 ± 0.55%), vascular wall-to-lumen ratio (0.63 ± 0.04 vs. 1.22 ± 0.08) and protein cast area (6.40 ± 0.07% vs. 20.27 ± 2.65%) were all significantly reduced in MMP9 KO rats (n=6) versus the corresponding values in SS rats (n=6) fed 8% HS diet for 3 weeks. Autoregulation of renal blood flow (RBF) to elevations in perfusion was impaired in SS rats prior to the development of hypertension, for RBF rose by 20.6 ± 3.6% (n=8) when MAP was increased from 110 to 150 mmHg. Autoregulation of RBF was restored in MMP9 KO rats and only increased by 7% when pressure was increased over the same range. In contrast, there was no difference in the fall in RBF in SS versus MMP9 KO rats when pressure was reduced from 110 to 50 mmHg. These findings suggest that knockout of MMP9 in SS rats restores autoregulation of RBF and opposes the development of hypertension, proteinuria, glomerular injury and renal interstitial fibrosis.

Inhibition of gelatinase B/MMP-9 does not attenuate colitis in murine models of inflammatory bowel disease

One third of patients with inflammatory bowel disease (IBD) inadequately respond to anti-TNF treatment and preclinical data suggest that matrix metalloproteinase-9 (MMP-9) is a novel therapeutic target. Here we show that IBD clinical and histopathological parameters found in wild type mice challenged with three different models of colitis, acute and chronic dextran sodium sulphate (DSS), and acute 2,4,6-trinitrobenzenesulfonic acid-induced colitis are not attenuated in MMP-9 knockout mice. We find similar colonic gene expression profiles in wild type and MMP-9 knockout mice in control and acute DSS conditions with the exception of eleven genes involved in antimicrobial response during colitis. Parameters of chronic colitis are similar in wild type and MMP-9 knockout mice. Pharmacological inhibition of MMP-9 with bio-active peptides does not improve DSS colitis. We suggest that MMP-9 upregulation is a consequence rather than a cause of intestinal inflammation and we question whether MMP-9 represents a disease target in IBD.

Matrix Metalloproteinase-9 and Synaptic Plasticity in the Central Amygdala in Control of Alcohol-Seeking Behavior

BackgroundDysfunction of the glutamatergic system has been implicated in alcohol addiction; however, the molecular underpinnings of this phenomenon are still poorly understood. In the current study we have investigated the possible function of matrix metalloproteinase-9 (MMP-9) in alcohol addiction because this protein has recently emerged as an important regulator of excitatory synaptic plasticity. MethodsFor long-term studies of alcohol drinking in mice we used IntelliCages. Dendritic spines were analyzed using Diolistic staining with DiI. Whole-cell patch clamp was used to assess silent synapses. Motivation for alcohol in human subjects was assessed on the basis of a Semi-Structured Assessment for the Genetics of Alcoholism interview. ResultsMice devoid of MMP-9 (MMP-9 knockout) drank as much alcohol as wild-type animals; however, they were impaired in alcohol seeking during the motivation test and withdrawal. The deficit could be rescued by overexpression of exogenous MMP-9 in the central nucleus of the amygdala (CeA). Furthermore, the impaired alcohol seeking was associated with structural alterations of dendritic spines in the CeA and, moreover, whole-cell patch clamp analysis of the basal amygdala to CeA projections showed that alcohol consumption and withdrawal were associated with generation of silent synapses. These plastic changes were impaired in MMP-9 knockout mice. Finally, C/T polymorphism of MMP-9 gene at position –1562, which upregulates MMP-9 expression, correlated with increased motivation for alcohol in alcoholics. ConclusionsIn aggregate, our results indicate a novel mechanism of alcohol craving that involves MMP-9–dependent synaptic plasticity in CeA.

Matrix metalloproteinase 9-dependent Notch signaling contributes to kidney fibrosis through peritubular endothelial-mesenchymal transition.

ABSTRACT Background: Endothelial cells are known to contribute to kidney fibrosis via endothelial–mesenchymal transition (EndoMT). Matrix metalloproteinase 9 (MMP-9) is known to be profibrotic. However, whether MMP-9 contributes to kidney fibrosis via EndoMT is unknown. Methods: Primary mouse renal peritubular endothelial cells (MRPECs) were isolated and treated by recombinant human transforming growth factor beta 1 (rhTGF-β1) with or without MMP-9 inhibitor or by recombinant human MMP-9 (rhMMP-9) alone. Kidney fibrosis was induced by unilateral ureteral obstruction (UUO) in MMP-9 knockout (KO) and wide-type (WT) control mice. The effects of MMP-9 on EndoMT of MRPECs and kidney fibrosis were examined. Results: We showed that MRPECs underwent EndoMT after rhTGF-β1 treatment or in UUO kidney as evidenced by decreased expression of endothelial markers, vascular endothelial cadherin (VE-cadherin) and CD31, and increased levels of mesenchymal markers, α-smooth muscle actin (α-SMA) and vimentin. The expression of fibrosis markers was also up-regulated significantly after rhTGF-β1 treatment in MRPECs. The EndoMT and fibrosis markers were significantly less in rhTGF-β1-treated MMP-9 KO MRPECs, whereas MMP-9 alone was sufficient to induce EndoMT in MRPECs. UUO kidney of MMP-9 KO mice showed significantly less interstitial fibrosis and EndoMT in MRPECs. Notch signaling shown by Notch intracellular domain (NICD) was increased, while Notch-1 was decreased in rhTGF-β1-treated MRPECs of MMP-9 WT but not MMP-9 KO mice. Inhibition of MMP-9 or Notch signaling prevented rhTGF-β1- or rhMMP-9-induced α-SMA and NICD upregulation in MRPECs. UUO kidney of MMP-9 KO mice had less staining of Notch signaling transcription factor Hey-1 in VE-cadherin-positive MRPECs than WT controls. Conclusions: Our results demonstrate that MMP-9-dependent Notch signaling plays an important role in kidney fibrosis through EndoMT of MRPECs.

Ablation of Matrix Metalloproteinase-9 Prevents Cardiomyocytes Contractile Dysfunction in Diabetics.

Elevated expression and activity of matrix metalloproteinase-9 (MMP9) and decreased contractility of cardiomyocytes are documented in diabetic hearts. However, it is unclear whether MMP is involved in the regulation of contractility of cardiomyocytes in diabetic hearts. In the present study, we tested the hypothesis that MMP9 regulates contractility of cardiomyocytes in diabetic hearts, and ablation of MMP9 prevents impaired contractility of cardiomyocytes in diabetic hearts. To determine the specific role of MMP9 in cardiomyocyte contractility, we used 12–14 week male WT (normoglycemic sibling of Akita), Akita, and Ins2+∕−/MMP9−∕− (DKO) mice. DKO mice were generated by cross-breeding male Ins2+∕− Akita (T1D) with female MMP9 knockout (MMP9−∕−) mice. We isolated cardiomyocytes from the heart of the above three groups of mice and measured their contractility and calcium transients. Moreover, we determined mRNA and protein levels of sarco-endoplasmic reticulum calcium ATPase-2a (SERCA-2a), which is involved in calcium handling during contractility of cardiomyocytes in WT, Akita, and DKO hearts using QPCR, Western blotting and immunoprecipitation, respectively. Our results revealed that in Akita hearts where increased expression and activity of MMP9 is reported, the rates of shortening and re-lengthening (±dL/dt) of cardiomyocytes were decreased, time to 90% peak height and baseline during contractility was increased, rate of calcium decay was increased, and calcium transient was decreased as compared to WT cardiomyocytes. However, these changes in Akita were blunted in DKO cardiomyocytes. The molecular analyses of SERCA-2a in the hearts showed that it was downregulated in Akita as compared to WT but was comparatively upregulated in DKO. These results suggest that abrogation of MMP9 gene prevents contractility of cardiomyocytes, possibly by increasing SERCA-2a and calcium transients. We conclude that MMP9 plays a crucial role in the regulation of contractility of cardiomyocytes in diabetic hearts.

Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction.

Matrix Metalloproteinase-2 (Mmp2) is a collagenase known to be important in the development of renal fibrosis. In unilateral ureteral obstruction (UUO) the obstructed kidney (OK) develops fibrosis, while the contralateral (CL) does not. In this study we investigated the effect of UUO on gene expression, fibrosis and pelvic remodeling in the kidneys of Mmp2 deficient mice (Mmp2-/-), heterozygous animals (Mmp2+/-) and wild-type mice (Mmp2+/+). Sham operated animals served as controls (Cntrl). UUO was prepared under isoflurane anaesthesia, and the animals were sacrificed after one week. UUO caused hydronephrosis, dilation of renal tubules, loss of parenchymal thickness, and fibrosis. Damage was most severe in Mmp2+/+ mice, while both Mmp2-/- and Mmp2+/- groups showed considerably milder hydronephrosis, no tubular necrosis, and less tubular dilation. Picrosirius red quantification of fibrous collagen showed 1.63±0.25% positivity in OK and 0.29±0.11% in CL (p<0.05) of Mmp2+/+, Mmp2-/- OK and Mmp2-/- CL exhibited only 0.49±0.09% and 0.23±0.04% (p<0.05) positivity, respectively. Mmp2+/- OK and Mmp2+/- CL showed 0.43±0.09% and 0.22±0.06% (p<0.05) positivity, respectively. Transcriptomic analysis showed that 26 genes (out of 48 examined) were differentially expressed by ANOVA (p<0.05). 25 genes were upregulated in Mmp2+/+ OK compared to Mmp2+/+ CL: Adamts1, -2, Col1a1, -2, -3a1, -4a1, -5a1, -5a2, Dcn, Fbln1, -5, Fmod, Fn1, Itga2, Loxl1, Mgp, Mmp2, -3, Nid1, Pdgfb, Spp1, Tgfb1, Timp2, Trf, Vim. In Mmp2-/- and Mmp2+/- 18 and 12 genes were expressed differentially between OK and CL, respectively. Only Mmp2 was differentially regulated when comparing Mmp2-/- OK and Mmp2+/- OK. Under stress, it appears that Mmp2+/- OK responds with less Mmp2 upregulation than Mmp2+/+ OK, suggesting that there is a threshold level of Mmp2 necessary for damage and fibrosis to occur. In conclusion, reduced Mmp2 expression during UUO protects mice against hydronephrosis and renal fibrosis.

Proteolytic regulation of synaptic plasticity in the mouse primary visual cortex: analysis of matrix metalloproteinase 9 deficient mice.

The extracellular matrix (ECM) is known to play important roles in regulating neuronal recovery from injury. The ECM can also impact physiological synaptic plasticity, although this process is less well understood. To understand the impact of the ECM on synaptic function and remodeling in vivo, we examined ECM composition and proteolysis in a well-established model of experience-dependent plasticity in the visual cortex. We describe a rapid change in ECM protein composition during Ocular Dominance Plasticity (ODP) in adolescent mice, and a loss of ECM remodeling in mice that lack the extracellular protease, matrix metalloproteinase-9 (MMP9). Loss of MMP9 also attenuated functional ODP following monocular deprivation (MD) and reduced excitatory synapse density and spine density in sensory cortex. While we observed no change in the morphology of existing dendritic spines, spine dynamics were altered, and MMP9 knock-out (KO) mice showed increased turnover of dendritic spines over a period of 2 days. We also analyzed the effects of MMP9 loss on microglia, as these cells are involved in extracellular remodeling and have been recently shown to be important for synaptic plasticity. MMP9 KO mice exhibited very limited changes in microglial morphology. Ultrastructural analysis, however, showed that the extracellular space surrounding microglia was increased, with concomitant increases in microglial inclusions, suggesting possible changes in microglial function in the absence of MMP9. Taken together, our results show that MMP9 contributes to ECM degradation, synaptic dynamics and sensory-evoked plasticity in the mouse visual cortex.

Matrix metalloproteinases-9 deficiency impairs liver regeneration through epidermal growth factor receptor signaling in partial hepatectomy mice

BackgroundLiver regeneration is a complex process regulated by many complex mechanisms involving cytokines, growth factors, metabolic networks, and so forth. Previous investigations have demonstrated that matrix metalloproteinase-9 (MMP-9) is an essential factor in liver regeneration. The present study aimed to explore the role of MMP-9 in epidermal growth factor receptor (EGFR) signaling and related proliferation signaling factors in a mouse partial hepatectomy (PH) model. Materials and methodsMMP-9 knockout (KO) and wild-type mice were used to establish the PH model. Liver regeneration was analyzed based on proliferation cell nuclear antigen immunohistochemistry and liver weight to body weight ratio. Also, EGFR ligands, EGFR, and downstream factors were measured by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot. ResultsMMP-9 KO mice showed a delayed hepatic regenerative response after PH. EGFR ligands, including heparin-binding epidermal growth factor and amphiregulin, were expressed at significantly lower levels between days 1 and 3 posthepatectomy in MMP-9 KO mice. MMP-9 KO mice also inhibited and delayed EGFR activation after PH. After PH, the expression of STAT3, NF-κB, and cyclinD1, all downstream of EGFR, was similar to EGFR activation. ConclusionsOur data provide new evidence supporting a critical role of MMP-9 in liver regeneration after PH through activation of EGFR signaling.

Matrix metalloproteinase 9 (MMP-9) is indispensable for long term potentiation in the central and basal but not in the lateral nucleus of the amygdala.

It has been shown that matrix metalloproteinase 9 (MMP-9) is required for synaptic plasticity, learning and memory. In particular, MMP-9 involvement in long-term potentiation (LTP, the model of synaptic plasticity) in the hippocampus and prefrontal cortex has previously been demonstrated. Recent data suggest the role of MMP-9 in amygdala-dependent learning and memory. Nothing is known, however, about its physiological correlates in the specific pathways in the amygdala. In the present study we show that LTP in the basal and central but not lateral amygdala (LA) is affected by MMP-9 knock-out. The MMP-9 dependency of LTP was confirmed in brain slices treated with a specific MMP-9 inhibitor. The results suggest that MMP-9 plays different roles in synaptic plasticity in different nuclei of the amygdala.