Increased Matrix Metalloproteinase Activity Research Articles

Molecular characterization of post-thrombotic syndrome

The post-thrombotic syndrome represents a poorly understood and significant vascular health problem. This review focuses on our current understanding of the pathogenesis of post-thrombotic syndrome. We emphasize the cellular and molecular mechanisms that are responsible for the critical components of post-thrombotic syndrome. These include the initiation of deep venous thrombosis, the pathogenesis of elevated venous pressure, and the factors responsible for nonhealing of venous stasis ulcers.

Preconditioning with Chronic Cerebral Hypoperfusion Reduces a Focal Cerebral Ischemic Injury and Increases Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 and Matrix Metalloproteinase-2 Expression

Atherosclerosis may cause severe stenosis of the arteries supplying the brain, which induces chronic cerebral hypoperfusion. Although an infarction often occurs in the chronically hypoperfused brain area, it has been uncertain whether the stroke severity is attenuated or increased when further decrease of blood flow occurs. To test the hypothesis that chronic cerebral hypoperfusion is protective against the subsequent severe ischemia, we examined the effect of chronic cerebral hypoperfusion on brains subjected to acute focal ischemia. Spontaneous hypertensive rats were subjected to middle cerebral artery occlusion/reperfusion four weeks after bilateral common carotid artery ligation (BCAL) or sham operation. The rats with BCAL had smaller infarctions, determined by 2,3,5-triphenyltetrazolium hydrochloride staining, and less severe neurologic deficits than those with sham operation. The number of DNA-damaged cells, examined by the in situ nick translation study, was significantly reduced in animals with BCAL. Immunoreactivity for apurinic/apyrimidinic endonuclease/redox factor-1, which plays a role in cellular defense mechanism, was markedly increased in those with BCAL. Indirect evidence of extracellular matrix remodeling, which might be associated with adaptive arteriogenesis or angiogenesis, was obtained in the form of increased matrix metalloproteinase-2 activity in them. These findings provide experimental evidence that chronic cerebral hypoperfusion would be protective against subsequent severe ischemic insults.

Significance and Therapeutic Potential of Endothelial Progenitor Cell Transplantation in a Cirrhotic Liver Rat Model

We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells. Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks. At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates. We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.

Increased Expression of Visfatin in Macrophages of Human Unstable Carotid and Coronary Atherosclerosis

Although the participation of inflammation in atherogenesis is widely recognized, the identification of the different components has not been clarified. In particular, the role of inflammation in plaque destabilization is not fully understood. Our main findings were as follows: (1) In a microarray experiment, we identified visfatin, one of the most recently identified adipokines, as a gene that was markedly enhanced in carotid plaques from symptomatic compared with plaques from asymptomatic individuals. This finding was confirmed when carotid plaques from 7 patients with asymptomatic and 14 patients with symptomatic lesions were examined with real-time reverse transcription polymerase chain reaction. (2) Immunohistochemistry showed that visfatin was localized in areas that were rich in lipid-loaded macrophages. (3) The relationship between visfatin and unstable lesions was also found in patients with coronary artery disease, demonstrating a strong visfatin immunostaining in lipid-rich regions within the material obtained at the site of plaque rupture in patients with acute myocardial infarction. (4) Both oxidized low-density lipoprotein and tumor necrosis factor-alpha increased visfatin expression in THP-1 monocytes, with a particularly enhancing effect when these stimuli were combined. (5) Visfatin increased matrix metalloproteinase-9 activity in THP-1 monocytes and tumor necrosis factor-alpha and interleukin-8 levels in peripheral blood mononuclear cells. Both of these effects were abolished when insulin receptor signaling was blocked. Our findings suggest that visfatin should be regarded as an inflammatory mediator, localized to foam cell macrophages within unstable atherosclerotic lesions, that potentially plays a role in plaque destabilization.

Can diesel particulate matter directly activate cardiac mast cells?

Rats subjected to inhaled diesel exhaust particles (DEP) developed a significant reduction in cardiac function. Histological examination of their hearts demonstrated a significant increase in cardiac mast cell (CMC) density coupled with increased matrix metalloproteinase activity. Pretreatment with a mast cell stabilizing compound prevented these effects on cardiac function indicating that they were mast cell-mediated. However, it was not clear if this mast cell activation was the result of a direct interaction of DEP with CMCs. The purpose of this study was to determine if DEP can directly activate CMCs. CMCs were isolated from rats and exposed to DEP or the mast cell secretagogue 48/80. 48/80 treatment triggered a significant histamine release above that of controls (80.5% and 26.1% of total histamine, respectively). However, exposure to DEP did not result in histamine release above that seen in controls (26.8%). Retained histamine levels were comparable for the control and DEP-exposed cells, ~50 ng/ml. These findings indicate that the DEP effects on cardiac function are not due to a direct action of DEP on CMCs. However, given the ability of endothelin-1 (ET-1) to stimulate mast cell activation in the whole heart and peritoneal mast cells, a DEP-induced ET-1 release from the lungs and subsequent paracrine activation of CMCs represents a plausible mechanism. Support: AHA 0435298N (JDG), EPA RD-83195301-0 (GLB)

Prostaglandin F2α and Its Receptor as Activators of Human Decidua

Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua, placenta, chorion, and amnion) are involved with all of the physiologies of parturition (membrane rupture, cervical dilatation, myometrial contractility, placental separation, and uterine involution). For parturition to occur, however, the intrauterine tissues need to first be activated to prepare for the work of labor, then stimulated to initiate labor. Prostaglandins normally are considered to be stimulators of the physiologies of labor. This review presents evidence that one prostaglandin, PGF2alpha, and its receptor, FP, are also activators, especially of the decidua. Stimulated by cytokines, the decidual synthesis of PGF2alpha and the expression of FP lead to increased matrix metalloproteinase activity, further enhancement of cytokine activity, increased decidual oxytocin and oxytocin receptor expression, decreased progesterone responsiveness, and possibly, enhanced expression of vascular endothelial growth factor. These collective actions prepare the decidua for its role in parturition.

Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.

IFN' synergizes with IL‐1α to up‐regulate MMP‐9 secretion in a cellular model of central nervous system tuberculosis

Matrix metalloproteinase-9 (MMP-9) activity is implicated in pathogenesis of central nervous system tuberculosis (CNS-TB). IFNgamma, a key cytokine in TB, usually inhibits MMP-9 secretion. Addition of IFNgamma to conditioned media from M. tb-infected monocytes (CoMTB) resulted in a 7-fold increase in MMP-9 activity detected by gelatin zymography (P<0.01). In contrast, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 secretion, measured by ELISA, was suppressed. Dexamethasone abolished the synergistic increase in MMP-9 activity. Interleukin (IL)-1beta in CoMTB is a critical mediator of synergy with IFNgamma, and IL-1beta alone synergizes with IFNgamma to increase MMP-9 secretion from 51 +/- 31 to 762 +/- 136 U. IL-1beta activity is dependent on p38 mitogen-activated protein (MAPK) kinase, which was found to be phosphorylated in tissue specimens from patients with CNS-TB. Extracellular signal regulated kinase (Erk) and p38 MAPK activation did not affect IFNgamma signaling pathways. Inhibition of janus-activated kinase (JAK)-2 by 50 microM AG540 decreased MMP-9 secretion to 124 +/- 11.1 from 651 +/- 229 U of activity (P<0.01). However, signal transducer and activator of transcription (STAT)-3 but not STAT-1 phosphorylation was synergistically up-regulated by IFNgamma and CoMTB. In summary, synergy between IL-1beta and STAT-3 dependent IFNgamma signaling is key in control of up-regulation of MMP-9 activity in CNS-TB and may be a significant mechanism of brain tissue destruction.

Effects of timed administration of doxycycline or methylprednisolone on post-myocardial infarction inflammation and left ventricular remodeling in the rat heart

The development of strategies to ameliorate post-myocardial infarction (MI) remodeling and improve function continues to be an area of clinical importance. Use of steroids for this purpose is controversial since the effects of timed treatment on relevant inflammatory, biochemical and structure/function endpoints are unclear. In a previous report, we demonstrated that use of doxycycline pre-treatment improves post-MI remodeling and passive left ventricular (LV) function. However, the effects of timed doxycycline post-MI treatment are unknown. To examine these issues, we performed a study using a rat MI model. Animals were administered one of the following: doxycycline (DOX), the corticosteroid methylprednisolone (MP), or aqueous vehicle. Treatment was given early, short-term (at time of MI to 24 h post-MI) or late, long term (2-7 days post-MI). Animals were sacrificed at 3, 7 or 42 days post-surgery. We assessed LV hemodynamics, pressure-volume, and pressure-scar strains, histomorphometry, inflammation via measurements of myeloperoxidase activity, and matrix metalloproteinase (MMP) activity. Late MP treatment yielded a robust right-shifted pressure-volume curve, which was accompanied by increased scar strains. Late DOX treatment yielded reduced average heart weight and size and preserved scar thickness. DOX treatment did not suppress inflammation, which contrasts with the suppressive effects of MP. Use of early or late MP yielded increased MMP activity in infarcted and non-infarcted regions. Early and late treatment with DOX yielded infarct-associated MMP activity levels comparable to those of vehicle-treated animals. In conclusion, results indicate that late use of MP yields adverse post-MI structure/function outcomes that correlate with suppression of inflammation and increased MMP activity. These observations contrast with those of DOX, in particular, late treatment where improved outcomes were observed in LV structure and were accompanied by the lack of suppression of inflammation.

Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-κB

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.

Prominent expression of metalloproteinases in early stages of ovarian tumorigenesis

The role for matrix metalloproteinases (MMPs) in tumor cells invasion and metastasis is well established, and expression of MMPs is recognized as an indication of tumor cell malignancy. Previous studies suggest that the degradation of the basement membrane is a crucial early step in epithelial transformation and ovarian tumorigenesis. Thus, MMPs may also express and exert a role in preneoplastic lesions of ovarian tissues. We investigated the expression of the major metalloproteinases, gelatinase A, 72 kDa type IV collagenase (MMP-2), and gelatinase B, 92 kDa type IV collagenase (MMP-9), and the presence of basement membrane in ovarian tumors and tissues from prophylactic oophorectomies using immunostaining. MMP expression was also characterized in a panel of ovarian cancer cell lines and several nontumorigenic ovarian surface epithelial primary cells by zymography, Northern, and Western blots. We found, surprisingly, that MMP-2 and MMP-9 are expressed more frequently in early lesions than in established carcinomas. No correlation was found between the expression of MMPs and tumor grades or stages. In preneoplastic lesions, MMP-2 or MMP-9 expression often associates with the absence of basement membrane and morphological alterations. MMP-2 is often expressed in nontumorigenic ovarian surface epithelial cells but reduced or absent in cancer cells. Thus, we conclude that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis and associates with the loss of epithelial basement membrane and morphological transformation. We propose that the increased MMP activity is an etiological factor for ovarian cancer risk. We found that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis. The finding suggests roles of MMP in tumor initiation in addition to invasion, and may impact on the strategy for use of MMP inhibitors in cancer prevention.

Infiltration of Inflammatory Cells Plays an Important Role in Matrix Metalloproteinase Expression and Activation in the Heart during Sepsis

Septicemia is an emerging pathological condition involving, among other effects, refractory hypotension and heart dysfunction. Here we have investigated the contribution of resident nonmyocytic cells to heart alterations after lipopolysaccharide administration. These cells contributed to the rapid infiltration of additional inflammatory cells that enhance the onset of heart disease through the release of inflammatory mediators. Early activation of resident monocytic cells played a relevant role on the infiltration process, mainly of major histocompatibility complex class II- and CD11b-positive cells. This infiltration was significantly impaired in animals lacking the nitric-oxide synthase-2 (NOS-2) gene or after pharmacological in-hibition of NOS-2 or cylooxygenase-2, suggesting a significant contribution of nitric oxide and prostanoids to the infiltration process. Under these conditions, the expression of NOS-2 and cylooxygenase-2 in the whole organ was attenuated because cardiomyocytes failed to express these enzymes. However, cardiomyocytes expressed and activated matrix metalloproteinase-9 through mechanisms regulated, at least in part, by nitric oxide and prostaglandins in an additive way. These results directly link the inflammatory response in the heart and extracellular matrix remodeling by the matrix metalloproteinases released by the cardiomyocytes, suggesting that activation and recruitment of inflammatory cells to the heart is a major early event in cardiac dysfunction promoted by septicemia.

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

Osteoclast differentiation is tightly regulated by receptor activator of NF-κB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to − 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to − 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from − 446 bp to − 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (− 1123 bp to − 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.

17β-Estradiol supplementation reduces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney

We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen alpha type IV [ND, 0.22 +/- 0.02; D, 0.99 +/- 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 +/- 0.08; D, 1.47 +/- 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen alpha type IV (D+E2, 0.47 +/- 0.10 ROD) and fibronectin (D+E2, 0.71 +/- 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 +/- 0.01; D, 0.62 +/- 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 +/- 0.02; D, 0.33 +/- 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 +/- 0.06; MMP-9, 0.73 +/- 0.01 ROD). The activities of MMP-2 (ND, 7.88 +/- 0.44; D, 5.60 +/- 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 +/- 1.8; D, 12.9 +/- 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 +/- 0.35; MMP-9, 21.4 +/- 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.

Inflammatory cells do not decrease the ultimate tensile strength of intact tendons in vivo and in vitro: protective role of mechanical loading

Although inflammatory cells and their products are involved in various pathological processes, a possible role in tendon dysfunction has never been convincingly confirmed and extensively investigated. The goal of this study was to determine whether or not an acute inflammatory process deprived of mechanical trauma can induce nonspecific damages to intact collagen fibers. To induce leukocyte accumulation, carrageenan was injected into rat Achilles tendons. We first tested the effect of leukocyte recruitment on the concentrations or activities of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Second, we analyzed at the biochemical, histological, and biomechanical levels the impact of leukocyte invasion on tendons. Finally, collagen bundles isolated from rat-tail tendons were exposed in vitro to mechanical stress and/or inflammatory cells to determine if mechanical loading could protect tendons from the leukocyte proteolytic activity. Carrageenan-induced leukocyte accumulation was associated with an increased matrix metalloproteinase activity and a decreased content of tissue inhibitors of matrix metalloproteinases. However, hydroxyproline content and load to failure did not change significantly in these tendons. Interestingly, mechanical stress, when applied in vitro, protected collagen bundles from inflammatory cell-induced deterioration. Together, our results suggest that acute inflammation does not induce damages to intact and mechanically stressed collagen fibers. This protective effect would not rely on increased tissue inhibitors of matrix metalloproteinases content but would rather be conferred to the intrinsic resistance of mechanically loaded collagen fibers to proteolytic degradation.

Toll-Like Receptor 9 Agonists Promote Cellular Invasion by Increasing Matrix Metalloproteinase Activity

Toll-like receptor 9 (TLR9) recognizes microbial DNA. We show here that TLR9 protein is expressed in human breast cancer cells and clinical breast cancer samples. Stimulation of TLR9-expressing breast cancer cells with the TLR9 agonistic CpG oligonucleotides (1-10 mumol/L) dramatically increased their in vitro invasion in both Matrigel assays and three-dimensional collagen cultures. Similar effects on invasion were seen in TLR9-expressing astrocytoma and glioblastoma cells and in the immortalized human breast epithelial cell line MCF-10A. This effect was not, however, dependent on the CpG content of the TLR9 ligands because the non-CpG oligonucleotides induced invasion of TLR9-expressing cells. CpG or non-CpG oligonucleotide-induced invasion in MDA-MB-231 cells was blunted by chloroquine and they did not induce invasion of TLR9(-) breast cancer cells. Treatment of MDA-MB-231 cells with CpG or non-CpG oligonucleotides induced the formation of approximately 50-kDa gelatinolytic band in zymograms. This band and the increased invasion were abolished by a matrix metalloproteinase (MMP) inhibitor GM6001 but not by a serine proteinase inhibitor aprotinin. Furthermore, CpG oligonucleotide treatment decreased tissue inhibitor of metalloproteinase-3 expression and increased levels of active MMP-13 in TLR9-expressing but not TLR9(-) breast cancer cells without affecting MMP-8. Neutralizing anti-MMP-13 antibodies inhibited the CpG oligonucleotide-induced invasion. These findings suggest that infections may promote cancer progression through a novel TLR9-mediated mechanism. They also propose a new molecular target for cancer therapy, because TLR9 has not been associated with cancer invasiveness previously.

Recruited leukocytes and local synthesis account for increased matrix metalloproteinase-9 activity in cerebrospinal fluid of dogs with central nervous system neoplasm

Matrix metalloproteinases (MMP) 2 and 9 are enzymes known to degrade several protein components of the extracellular matrix. In humans, increased concentrations of these enzymes have been demonstrated in the cerebrospinal fluid (CSF) of subjects affected by many neurological conditions including brain tumours; nevertheless comparative data in dogs are completely lacking. Aim of this study was to investigate these molecules in CSF of dogs diagnosed with CNS neurological diseases. Higher activity of MMP 2 and 9 was revealed in dogs with space occupying lesions of likely neoplastic origin in comparison to dogs with idiopathic epilepsy. Statistical modelling reveals that increased MMP 9 activity is mainly due to leukocytes recruitment and local synthesis.

Interleukin-6 Upregulates Expression of KDR and Stimulates Proliferation of Human Cerebrovascular Smooth Muscle Cells

Interleukin-6 (IL-6) may play multiple roles in angiogenesis and vascular remodeling. Our previous study showed that a promoter polymorphism (174G>C) in IL-6 is associated with brain arteriovenous malformation hemorrhage; tissue expression is related to genotype. In this study, we investigated the effects of IL-6 on human cerebral smooth muscle cells (HCSMCs) and smooth muscle cells isolated from brain arteriovenous malformation surgical specimens (AVM SMCs) and surgical controls (control HCSMCs--from structurally normal temporal lobe taken during surgical treatment of epilepsy patients). We found that IL-6 (1.1+/-0.27 versus 0.37+/-0.04 pg/mL, n=5, P<0.05) and endogenous vascular endothelial growth factor (VEGF) receptor II (kinase domain-containing receptor (KDR), 15+/-3 versus 1.5+/-3 pg/mL, n=5, P<0.05) were increased in brain AVM SMCs compared with control HCSMCs. Further research revealed that IL-6 could stimulate SMC proliferation, VEGF release, and KDR activation in control HCSMCs. It could also stimulate KDR phosphorylation in control HCSMCs, further confirming a unique role of IL-6 in the triggering of KDR. Interleukin-6 could increase matrix metalloproteinase-9 (MMP-9) secretion through activating KDR in control HCSMCs (P<0.05 versus control). Inhibiting IL-6-induced KDR could reduce MMP-9 activity at least 50% compared with the control group (P<0.05). Increased MMP-9 activity was accompanied by increased control HCSMC proliferation, and blocking MMP-9 activity significantly reduced IL-6-induced control HCSMC proliferation (P<0.05). Collectively, our results show that IL-6 could activate, amplify, and maintain the angiogenic cascade in HCSMCs. A novel role of IL-6 during HCSMC proliferation is upregulating KDR expression and phosphorylation. The results may contribute to the angiogenic phenotype of human brain vascular diseases, such as brain AVM.

In vitro inhibition of matrix metalloproteinase activity in tracheal epithelial lining fluid from horses with recurrent airway obstruction

To evaluate inhibitory effects of synthetic matrix metalloproteinase (MMP) inhibitors in vitro on gelatinolytic and collagenolytic activities in tracheal epithelial lining fluid (TELF) of horses with recurrent airway obstruction (RAO). 10 horses with RAO and 5 healthy control horses. Substrate-based functional assays, collagen I and gelatin degradation, were used to measure endogenous collagenolytic and gelatinolytic activities in TELF. In vitro inhibition of MMP activity in TELF with 2 chemically modified tetracyclines (CMTs; CMT-3 and CMT-8) and 2 bisphosphonates (BPs; zoledronate and pamidronate) was evaluated. CMT-3, CMT-8, zoledronate, and pamidronate in a dose-dependent manner inhibited TELF type I collagenolytic and gelatinolytic activities, although no complete inhibition of TELF type I collagenolytic and gelatinolytic activities was achieved with the inhibitor concentrations of 25 to 500 microM tested. The CMTs inhibited pathologically induced collagen I degradation more effectively than BPs. Of the tested CMTs, CMT-3 was the most effective inhibitor of gelatinolytic activity, and the efficiency of CMT-3 corresponded with that of the BPs. An increase in MMP activity in the equine respiratory tract may potentially be inhibited by administration of CMTs or BPs. Distinct synthetic MMP inhibitors may eventually provide an additional means for pharmacologic treatment by decreasing ongoing active tissue destructive inflammation associated with chronic lung disease. The MMP inhibitors such as CMTs and BPs that are targeted to solely inhibit a pathologic increase in MMP activities provide the advantage of minimal adverse effects that are characteristics of other excessively potent MMP inhibitors.

Hypercapnic Emphysema in C3H Mice Induced by Cigarette Smoke (CS) Demonstrates Pulmonary Inflammation Concomitant With Increased Matrix Metalloproteinases (MMPs) and Substance P (SP)

We have recently established a model of CS-induced emphysema in C3H mice characterized by hypercapnia and depressed ventilatory response that closely resembles the major features of patients with hypercapnic chronic obstructive pulmonary disease (COPD). COPD is often associated with pulmonary inflammation. Previous studies have shown that increased MMP activity, IL-1β and SP are involved in inflammatory process, and the former two may be involved in pulmonary parenchymal destruction associated with emphysema and the latter is implicated in airway remodeling. Therefore, we asked whether this animal model has pulmonary inflammation, and if so, whether MMPs, IL-1β, SP and its receptor (neurokinin A receptor, NK1R) were altered. The mice were exposed to CS (100 mg TPM/m3 in the first wk and 250 mg for the following wks) or filtered air for 16 wks. After exposure, pulmonary MMP- 2, 9, and 12 were detected by RT-PCR and the activity of MMP-2 and-9 was assessed by zymography, IL-1β was measured by ELISA, and SP and NK1R were analyzed by EIA and RT-PCR, respectively. We found that CS-induced emphysema was associated with pulmonary inflammation characterized by infiltrates of macrophages, neutrophils and lymphocytes. CS also significantly upregulated: (a) SP level and expression of NK1 mRNA; (b) MMP2, 9 and 12 mRNA expressions and MMP-2 and -9 enzymatic activity; and (c) IL-1β in lung tissues. Our data confirm the pulmonary inflammation and suggest that the observed elevation of MMPs, IL-1β, SP and NK1R may be involved in pathogenesis of emphysema in this model. (Supported by ALA CI-016N and NIH 74183)