Effects Of Matrix Metalloproteinases Inhibition Research Articles

Intravital Microscopy to Study the Effect of Matrix Metalloproteinase Inhibition on Acute Myeloid Leukemia Cell Migration in the Bone Marrow.

Hematopoiesis is the process through which all mature blood cells are formed and takes place in the bone marrow (BM). Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. AML progression causes drastic remodeling of the BM microenvironment, making it no longer supportive of healthy hematopoiesis and leading to clinical cytopenia in patients. Understanding the mechanisms by which AML cells shape the BM to their benefit would lead to the development of new therapeutic strategies. While the role of extracellular matrix (ECM) in solid cancer has been extensively studied during decades, its role in the BM and in leukemia progression has only begun to be acknowledged. In this context, intravital microscopy (IVM) gives the unique insight of direct in vivo observation of AML cell behavior in their environment during disease progression and/or upon drug treatments. Here we describe our protocol for visualizing and analyzing MLL-AF9 AML cell dynamics upon systemic inhibition of matrix metalloproteinases (MMP), combining confocal and two-photon microscopy and focusing on cell migration.

The pathogenesis of aging-induced left atrial appendage thrombus formation and cardioembolic stroke in mice is influenced by inflammation-derived matrix metalloproteinases

Elderly people without atrial fibrillation (AF) still have a high incidence of cardioembolic stroke, suggesting that thrombus formation within the left atrial appendage (LAA) may also occur in an AF-independent manner. In the present study, we explored the potential mechanisms for aging-induced LAA thrombus formation and stroke in mice. We monitored stroke events in 180 aging male mice (14–24 months) and assessed left atrium (LA) remodeling by echocardiography at different ages. Mice that had stroke were implanted with telemeters to confirm AF. Histological features of LA and LAA thrombi were examined, as well as collagen content, expression of matrix metalloproteinases (MMPs), and leukocyte density in the atria at different ages, in mice with or without stroke. Also, the effects of MMP inhibition on stroke incidence and atrial inflammation were tested. We detected 20 mice (11 %) with stroke, 60 % of which were within 18–19 months of age. Although we did not detect AF in mice with stroke, we detected the presence of LAA thrombi, suggesting that stroke originated from the hearts of these mice. Compared with 18-month-old mice without stroke, 18-month-old stroke mice had enlarged LA with a very thin endocardium, that was associated with less collagen and heightened MMP expression in the atria. During aging, we found that the expression of mRNAs for atrial MMP7, MMP8, and MMP9 peaked at 18 months, which closely correlated with reductions in collagen content and the time-window for cardioembolic stroke in these mice. Treatment of mice with an MMP inhibitor at 17–18 months of age reduced atrial inflammation and remodeling, and stroke incidence. Taken together, our study demonstrates that aging-induced LAA thrombus formation occurs through a mechanism involving upregulation of MMPs and breakdown of collagen, and that treatment with an MMP inhibitor may be effective as a treatment strategy for this heart condition.

Anti-Metastatic and Anti-Inflammatory Effects of Matrix Metalloproteinase Inhibition by Ginsenosides.

Matrix metalloproteinases (MMPs) are proteolytic enzymes which cleave extracellular matrix (ECM) and other substrates. They are deeply involved in both cancer metastasis and human chronic inflammatory diseases such as osteoarthritis and Crohn’s disease. Regulation of MMPs is closely associated with signaling molecules, especially mitogen-activated protein kinases (MAPKs), including three representative kinases, extracellular signal regulated kinases (ERK), p38 and c-Jun N-terminal kinases (JNK). Ginseng (Panax sp.) is a plant which has been traditionally used for medicinal applications. Ginsenosides are major metabolites which have potentials to treat various human diseases. In this review, the pharmacological effects of ginsenosides have been rigorously investigated; these include anti-metastatic and anti-inflammatory activities of ginsenosides associated with suppression of MMPs via regulation of various signaling pathways. This will highlight the importance of MMPs as therapeutic targets for anti-metastatic and anti-inflammatory drug development based on ginsenosides.

Effects of Matrix Metalloproteinases on the Performance of Platelet Fibrin Gel Spiked With Cardiac Stem Cells in Heart Repair.

Stem cells and biomaterials have been studied for therapeutic cardiac repair. Previous studies have shown the beneficial effects of platelet fibrin gel and cardiac stem cells when cotransplanted into rodent hearts with myocardial infarction (MI). We hypothesized that matrix metalloproteinases (MMPs) play an important role in such protection. Thus, the present study is designed to elucidate the effects of MMP inhibition on the therapeutic benefits of intramyocardial injection of platelet fibrin gel spiked with cardiac stem cells (cell-gel) in a rat model of acute MI. In vitro, broad-spectrum MMP inhibitor GM6001 undermines cell spreading and cardiomyocyte contraction. In a syngeneic rat model of myocardial infarction, MMP inhibition blunted the recruitment of endogenous cardiovascular cells into the injected biomaterials, therefore hindering de novo angiogenesis and cardiomyogenesis. Echocardiography and histology 3 weeks after treatment revealed that metalloproteinase inhibition diminished the functional and structural benefits of cell-gel in treating MI. Reduction of host angiogenesis, cardiomyocyte cycling, and MMP-2 activities was evident in animals treated with GM6001. Our findings suggest that MMPs play a critical role in the therapeutic benefits of platelet fibrin gel spiked with cardiac stem cells for treating MI. In this study, the effects of matrix metalloproteinase inhibition on the performance of platelet gel spiked with cardiac stem cells (cell-gel) for heart regeneration are explored. The results demonstrate that matrix metalloproteinases are required for cell-gel to exert its benefits in cardiac repair. Inhibition of matrix metalloproteinases reduces cell engraftment, host angiogenesis, and recruitment of endogenous cardiovascular cells in rats with heart attack.

Abstract 14680: Evaluation of the Protective Effects of Long-term Doxycycline Treatment on Progression of Marfan-associated Aortic Aneurysm by High-resolution Ultrasound Imaging

Introduction: Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in fibrillin-1 gene. We have previously reported that doxycycline, an antibiotic and a general inhibitor of matrix metalloproteinases (MMPs), could improve aortic contractility and elastin fibers structure and organization in a mouse model of MFS. However, the in vivo effects of MMPs inhibition on the progression of aneurysm and pulse wave velocity (PWV) have not been investigated. In this study, we assessed the hypothesis that a long-term treatment with a sub-antibiotic dose of doxycycline would delay the progression of aortic aneurysm and improve the PWV in the Marfan mouse. Methods: In this study, we have used a high-resolution, high-contrast ultrasound imaging technique to conduct a longitudinal study in order to measure aortic diameter (dilatation) and PWV (indication of aortic elasticity/stiffness) in the animals. Marfan (n=12) and control (n=13) mice were given a sub-antibiotic dose of doxycycline in their drinking water at a concentration of 0.24 g/L/day beginning at 6 weeks of age, whereas another group of Marfan (n=12) and control (n=12) mice were given plain drinking water for comparison. At 3, and 6 months of age, aortic diameter and PWV of the experimental animals were measured by ultrasound imaging. Results: At 3 months of age, measurement of aortic root diameter showed significant dilatation in both treated and non-treated Marfan mice as compared to control groups (p < 0.01). However, at 6 months of age, the aortic root diameter in treated Marfan mice was not different from those in controls. The same observation was made for the Sinus of Valsalva region. Especially in this region, treated Marfan aorta showed significantly reduced diameter compared to non-treated Marfan groups (p < 0.05). With respect to the measurement of PWV, a significant increase was observed in 3- and 6-month Marfan mice aorta vs. control (p < 0.01). Interestingly, doxycycline treatment normalized the PWV in 6-month old Marfan mice compared to non-treated Marfan aorta. Conclusions: Our findings show that long-term doxycycline treatment has protective effects in terms of reduction of aortic dilatation and stiffness in Marfan mice.

Postresuscitation Administration of Doxycycline Preserves Cardiac Contractile Function in Hypoxia-Reoxygenation Injury of Newborn Piglets*

Cardiac injury is common in asphyxiated neonates and is associated with matrix metalloproteinase-2 activation. Although studies have demonstrated the cardioprotective effects of matrix metalloproteinase inhibition, this has not been tested in clinically translatable models of hypoxia-reoxygenation injury. We aimed to elucidate the effect of doxycycline, a matrix metalloproteinase inhibitor, on cardiac injury and functional recovery in a swine model of neonatal hypoxia-reoxygenation. Thirty-three newborn piglets were acutely instrumented for continuous monitoring of cardiac output and systemic arterial pressure. After stabilization, normocapnic alveolar hypoxia (10-15% oxygen) was instituted for 2 hours followed by 4 hours of normoxic reoxygenation. Piglets were blindly, block randomized to receive IV boluses of normal saline (control) and doxycycline at 5 minutes of reoxygenation (n = 7/group). Sham-operated piglets (n = 5) received no hypoxia-reoxygenation. Markers of myocardial injury (plasma and myocardial tissue troponin I; myocardial lactate) and oxidative stress (lipid hydroperoxides) were measured by enzyme-linked immunosorbent assay and Western blot. Myocardial matrix metalloproteinase-2 activity was quantified by gelatin zymography and immunoprecipitation. University animal laboratory. Piglets (1-4 d old, weighing 1.4-2.5 kg). IV doxycycline (3, 10, or 30 mg/kg) given during resuscitation. Hypoxic piglets had cardiogenic shock (cardiac output 58% ± 1% of baseline), hypotension (systemic arterial pressure 31 ± 1 mm Hg), and acidosis (pH 7.02 ± 0.02). Doxycycline improved cardiac and stroke volume index with no chronotropic effect in doxycycline-treated piglets compared with controls. Systemic arterial pressure was higher and the pulmonary artery pressure/systemic arterial pressure ratio was lower in doxycycline groups, with reduced levels of markers of myocardial injury and oxidative stress in doxycycline-treated piglets compared with controls. Negative correlations were found between markers of myocardial injury (plasma troponin I, myocardial lactate) and functional recovery and between myocardial tissue and plasma troponin I. Doxycycline-treated piglets had lower myocardial matrix metalloproteinase-2 activity compared with controls. Postresuscitation administration of doxycycline attenuates cardiac injury and improves functional recovery in newborn piglets with hypoxia-reoxygenation.

MT5-MMP, ADAM-10, and N-Cadherin Act in Concert To Facilitate Synapse Reorganization after Traumatic Brain Injury

Matrix metalloproteinases (MMPs) influence synaptic recovery following traumatic brain injury (TBI). Membrane type 5-matrix metalloproteinase (MT5-MMP) and a distintegrin and metalloproteinase-10 (ADAM-10) are membrane-bound MMPs that cleave N-cadherin, a protein critical to synapse stabilization. This study examined protein and mRNA expression of MT5-MMP, ADAM-10, and N-cadherin after TBI, contrasting adaptive and maladaptive synaptogenesis. The effect of MMP inhibition on MT5-MMP, ADAM-10, and N-cadherin was assessed during maladaptive plasticity and correlated with synaptic function. Rats were subjected to adaptive unilateral entorhinal cortical lesion (UEC) or maladaptive fluid percussion TBI+bilateral entorhinal cortical lesion (TBI+BEC). Hippocampal MT5-MMP and ADAM-10 protein was significantly elevated 2 and 7 days post-injury. At 15 days after UEC, each MMP returned to control level, while TBI+BEC ADAM-10 remained elevated. At 2 and 7 days, N-cadherin protein was below control. By the 15-day synapse stabilization phase, UEC N-cadherin rose above control, a shift not seen for TBI+BEC. At 7 days, increased TBI+BEC ADAM-10 transcript correlated with protein elevation. UEC ADAM-10 mRNA did not change, and no differences in MT5-MMP or N-cadherin mRNA were detected. Confocal imaging showed MT5-MMP, ADAM-10, and N-cadherin localization within reactive astrocytes. MMP inhibition attenuated ADAM-10 protein 15 days after TBI+BEC and increased N-cadherin. This inhibition partially restored long-term potentiation induction, but did not affect paired-pulse facilitation. Our results confirm time- and injury-dependent expression of MT5-MMP, ADAM-10, and N-cadherin during reactive synaptogenesis. Persistent ADAM-10 expression was correlated with attenuated N-cadherin level and reduced functional recovery. MMP inhibition shifted ADAM-10 and N-cadherin toward adaptive expression and improved synaptic function.

Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCs cultured in the presence of adhesive substrates.

Cell adhesion to extracellular matrix (ECM) generates intracellular signals that modulate cell survival, proliferation, migration and differentiation. Because of its heterogeneous nature, ECM has the potential to induce unique responses that are composition-dependent. One approach to study the effect of ECM signals on cell development, independently on signals from other extracellular sources, has been to deprive cells of serum and to analyze the influence of specific ligands. In the current work we determine the potential of different ECM proteins (fibronectin, laminin, collagen) on the proliferation and differentiation of human umbilical cord blood-derived neural stem cells (HUCB-NSCs) cultured in serum-free conditions. The effect of tested ECM components on the above processes might be associated with the particular pattern of their proteolysis. In this context enzymes that are responsible for the modification of ECM proteins are of particular pertinence. Matrix metalloproteinases (MMPs) represent a family of enzymes known to play role in the modification of ECM and by this can change the cell-ECM substrate interaction, required for cell development. In an effort to elucidate the participation of MMPs in the proliferation and differentiation HUCB-NSCs were cultured in the presence or absence of MMPs inhibitors - GM6001 and doxycycline. Our results show that addition of the above inhibitors interfered with both the proliferation and differentiation of progenitor cells toward the neuronal lineage. This effect depends on the adhesive ECM substrate and is most pronounced in the presence of fibronectin and laminin. In conclusion, our results suggest that MMPs modulate interaction between HUCB-NSCs and their environment and therefore might be an important component in neurogenesis-associated processes.

The effect of matrix metalloproteinase inhibition on tendon-to-bone healing in a rotator cuff repair model

Recent studies have demonstrated a potentially critical role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathophysiology of rotator cuff tears. We hypothesize that local delivery of a MMP inhibitor after surgical repair of the rotator cuff will improve healing at the tendon-to-bone surface interface. Sixty-two male Sprague-Dawley rats underwent acute supraspinatus detachment and repair. In the control group (n=31), the supraspinatus was repaired to its anatomic footprint. In the experimental group (n=31), recombinant alpha-2-macroglobulin (A2M) protein, a universal MMP inhibitor, was applied at the tendon-bone interface with an identical surgical repair. Animals were sacrificed at 2 and 4 weeks for histomorphometry, immunohistochemistry, and biomechanical testing. Statistical comparisons were performed using unpaired t tests. Significance was set at P < .05. Significantly greater fibrocartilage was seen at the healing enthesis in the A2M-treated specimens compared with controls at 2 weeks (P < .05). Significantly greater collagen organization was observed in the A2M-treated animals compared with controls at 4 weeks (P < .01). A significant reduction in collagen degradation was observed at both 2 and 4 weeks in the experimental group (P < .05). Biomechanical testing revealed no significant differences in stiffness or ultimate load-to-failure. Local delivery of an MMP inhibitor is associated with distinct histologic differences at the tendon-to-bone interface after rotator cuff repair. Modulation of MMP activity after rotator cuff repair may offer a novel biologic pathway to augment tendon-to-bone healing after rotator cuff repair.

Early Beneficial Effect of Matrix Metalloproteinase Inhibition on Blood—Brain Barrier Permeability as Measured by Magnetic Resonance Imaging Countered by Impaired Long-Term Recovery after Stroke in Rat Brain

Proteolytic disruption of the extracellular matrix with opening of the blood-brain barrier (BBB) because of matrix metalloproteinases (MMPs) occurs in reperfusion injury after stroke. Matrix metalloproteinase inhibition blocks the early disruption of the BBB, but the long-term consequences of short-term MMP inhibition are not known. Recently, a method to quantify BBB permeability by graphical methods was described, which provides a way to study both early disruption of the BBB and long-term effects on recovery in the same animal. We used a broad-spectrum MMP inhibitor, BB1101, to determine both the usefulness of the Magnetic resonance imaging (MRI) method for treatment studies and the long-term effects on recovery. Magnetic resonance imaging studies were performed in control (N=6) and drug-treated (N=8) groups on a dedicated 4.7-T MRI scanner. Adult Wistar-Kyoto underwent a 2-h middle cerebral artery occlusion followed by an MRI study after 3 h of reperfusion, which consisted of T2- and diffusion-weighted techniques. Additionally, a rapid T1 mapping protocol was also implemented to acquire one pre-gadolinium-diethylenetriaminepentaacetic acid baseline data set followed by postinjection data sets at 3-min intervals for 45 mins. The same animal was imaged again at 48 h for lesion size estimation. Data was postprocessed pixel-wise to generate apparent diffusion coefficient and permeability coefficient maps. Treatment with BB-1101 significantly reduced BBB permeability at 3 h, but failed to reduce lesion size at 48 h. Behavioral studies showed impairment in recovery in treated rats. Magnetic resonance imaging allowed for the monitoring of multiple parameters in the same animal. Our studies showed that BB-1101 was an excellent inhibitor of the BBB damage. However, results show that BB-1101 may be responsible for significant deterioration in neurologic status of treated animals. Although these preliminary results suggest that BB-1101 is useful in reducing early BBB leakage owing to reperfusion injury in stroke, further studies will be needed to determine whether the later detrimental effects can be eliminated by shorter time course of drug delivery.

Effects of matrix metalloproteinase inhibition on short‐ and long‐term plasticity of schaffer collateral/CA1 synapses

It is increasingly evident that matrix metalloproteinases (MMPs), a family of zinc containing extracellular endopeptidases, participate in processes supporting hippocampal synaptic plasticity. The purpose of this study was to further the understanding of MMPs involvement in hippocampal plasticity. Acute hippocampal slices, generated from 20- to 30-day-old male Sprague-Dawley rats, were subjected to various electrophysiologic stimulatory paradigms to produce either short-term or long-term modifications to synaptic efficacy. Slices exposed to broad-spectrum MMP inhibitor, FN-439, exhibited impairments in paired-pulse facilitation, theta-burst facilitation, and long-term depression. Additionally, we observed that MMP inhibition impaired both the induction and stability of long-term potentiation (LTP). Furthermore, evidence indicated that the effect of MMP inhibition on LTP maintenance is dependent upon integrin-directed adhesion, whereas the effects of MMP inhibition on LTP induction are independent of integrin-directed adhesion. Together, these data support a generalized role for MMPs in short-term and long-term hippocampal plasticity and indicate that MMPs are a necessary facet of integrin-mediated cell adhesion supporting LTP stabilization.

Metalloproteinase Inhibition Has Differential Effects on Alloimmunity, Autoimmunity, and Histopathology in the Transplanted Lung

Upregulation of matrix metalloproteinases (MMPs) has been associated with chronic lung allograft rejection known as bronchiolitis obliterans syndrome. It has been suggested that MMP inhibition could prevent the rejection response. However, the effect of MMP inhibition on lung allograft rejection has not been reported. Utilizing a rat model of lung transplantation, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were overexpressed by gene therapy in F344 rat lung allografts prior to transplantation into WKY recipient rats. Separately, WKY rats that received F344 lung allografts were treated systemically with COL-3, a global MMP inhibitor. TIMP-1 and TIMP-2 had differential effects on delayed type hypersensitivity (DTH) responses to donor antigens and type V collagen, an autoantigen involved in the rejection response. Neither TIMP-1 or TIMP-2 affected the onset of rejection pathology. COL-3 suppressed DTH responses to donor antigens and type V collagen, abrogated local production of tumor necrosis factor-alpha, and interleukin-1beta. Although it did not prevent rejection pathology, COL-3 (30 mg/kg) induced intragraft B cell hyperplasia suggestive of posttransplant proliferative disorder (PTLD). These data identify a complex role for MMPs and TIMPs in the immunopathogenesis of lung allograft rejection, and indicate their effects are not limited to matrix remodeling.

Effect of Matrix Metalloproteinase Inhibition by Doxycycline on Myocardial Healing and Remodeling after Myocardial Infarction

The aim of conducting this study was to assess the clinical relevance of matrix metalloproteinase (MMP) inhibition by doxycycline, an effective MMP inhibitor, in a rat model of extensive myocardial infarction (MI) and left ventricular (LV) dysfunction. Rats (n = 22) were subjected to extensive anterior MI. Doxycycline (25 mg SC, daily) or saline (control) injections were started for nine days thereafter. The effect of doxycycline on MMP activity in the infarcted and remote myocardium was measured by zymography, in another subgroup (n = 8), nine days after MI. Echocardiography and magnetic resonance imaging (MRI) studies were performed at one and thirty days after MI to assess LV remodeling and function. After 4 weeks, hearts were fixed, and subjected to morphometric and histological analysis. Compared with control, doxycycline treatment attenuated MMP-9 and -2 activity in both infarcted and remote myocardium. Serial echocardiography studies showed that doxycycline failed to attenuate scar thinning, LV dilatation and dysfunction. MRI study showed that doxycycline impaired LV compensatory hypertrophy. Furthermore, compared with control, doxycycline reduced vessel density (/mm(2) +/- SEM) in the infarcted myocardium (84 +/- 16 vs. 46 +/- 9/mm(2), respectively; p < 0.05). Our work suggest that effective MMPs' inhibition in the infarcted and remote myocardium by doxycycline does not prevent LV remodeling and dysfunction but impairs angiogenesis and compensatory LV hypertrophy. Our findings caution against aggressive, non-selective inhibition of MMPs in the early healing phase after MI.

Effect of matrix metalloproteinase inhibition on adipose tissue development

The effect of Ro 28-2653, a synthetic matrix metalloproteinase (MMP) inhibitor, on adipose tissue development was studied in mice kept on a high fat diet (HFD). Five-week-old male wild-type (C57Bl/6J) mice were fed the HFD (42% kcal as fat, 20.1 kJ/g) and received daily p.o. instillations of inhibitor (30 mg/kg) or vehicle. After 15 weeks of the HFD, the body weight gain was lower in the inhibitor-treated group (7.4 ± 0.88 g versus 10 ± 1.4 g) whereas the weights of the isolated subcutaneous (SC) or gonadal (GON) fat deposits were 10–15% lower. The number of adipocytes in adipose tissues of the inhibitor-treated mice was somewhat higher (10–17%) but their diameter was smaller (about 10%). In situ zymography showed reduced gelatinolytic activity in SC (about 2.7-fold) and GON (1.4-fold) adipose tissue of inhibitor-treated mice, whereas their fibrillar collagen content was higher (1.5- and 4.7-fold, respectively). In both SC and GON adipose tissues of inhibitor-treated mice, MMP-2 (gelatinase A) and MMP-14 (membrane type-1 MMP) were 2- to 3-fold upregulated, whereas MMP-9 (gelatinase B) mRNA levels were not affected. Thus, in this in vivo model partial inhibition of gelatinolytic activity is associated with moderate effects on adipose tissue development and cellularity. Possibly, enhanced MMP expression to some extent counteracts the in vivo effect of the inhibitor in adipose tissue.

Matrix metalloproteinase inhibition modifies left ventricular remodeling after myocardial infarction in pigs

Background: Global and regional shape changes that occur within the left ventricular wall after myocardial infarction have been termed infarct expansion. A potential mechanism for this postinfarction remodeling is activation of the matrix metalloproteinases. Accordingly, the present study examined the effects of matrix metalloproteinase inhibition on left ventricular global geometry after myocardial infarction in pigs. Methods: Myocardial infarction was created in pigs by means of occlusion of the first and second obtuse marginal branches of the circumflex coronary artery, resulting in a uniform left ventricular free wall infarct size of 21% ± 2%. At 5 days after infarction, the pigs were randomized to undergo broad-spectrum matrix metalloproteinase inhibition (n = 9; PD166793, 20 mg · kg−1 · d−1 by mouth) or myocardial infarction alone (n = 8). Ten pigs served as noninfarction control animals. Left ventricular end-diastolic area, determined by means of echocardiography, was measured 8 weeks after infarction. Results: Left ventricular end-diastolic area increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and myocardial infarction only groups compared to reference control animals (3.7 ± 0.2 cm2), but was reduced with broad-spectrum matrix metalloproteinase inhibition compared to myocardial infarction alone (4.5 ± 0.2 vs 4.9 ± 0.2 cm2, respectively; P <.05). Regional radial stress within the infarct region increased in both infarction groups when compared to values obtained from reference control animals (599 ± 152 g/cm2), but was attenuated in the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition group compared to the myocardial infarction alone group (663 ± 108 vs 1242 ± 251 g/cm2, respectively; P <.05). Similarly, regional myocardial stiffness increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and the myocardial infarction only groups compared with that observed in reference control animals (14 ± 1 rkm, P <.05) but was lower with broad-spectrum matrix metalloproteinase inhibition than with myocardial infarction alone (42 ± 6 vs 68 ± 10 rkm, respectively; P <.05). Conclusions: Matrix metalloproteinase inhibition reduced postinfarction left ventricular dilation, reduced regional myocardial wall stress, and modified myocardial material properties. These unique findings suggest that increased myocardial matrix metalloproteinase activation after infarction contributes directly to the left ventricular remodeling process.J Thorac Cardiovasc Surg 2003;125:602-10

Effects of Matrix Metalloproteinase Inhibition on Ventricular Remodeling Due to Volume Overload

Left ventricular (LV) hypertrophy and dilatation are important compensatory responses to chronic volume overload. Although LV function is initially preserved by these responses, the continued structural remodeling of the myocardium ultimately becomes maladaptive, leading to the development of heart failure. We have shown previously that increased myocardial matrix metalloproteinase (MMP) activity precedes LV dilatation induced by a chronic volume overload. Accordingly, this study focused on the effects of MMP inhibition therapy (PD 166793, 1 mg x kg(-1) x d(-1)) on LV size and function in a rat model of volume overload-induced heart failure. Rats were divided into the following groups: treated and untreated infrarenal abdominal aortocaval fistula and treated and untreated sham-operated (control). LV weights of both fistula groups were increased above that of the control group (868+/-79 mg; P< or =0.001); LV weights in the treated fistula group, however, were lower than in the untreated fistula group at 8 weeks (1447+/-186 versus 1715+/-279 mg, respectively; P< or =0.012). The marked ventricular dilatation seen in the untreated fistula group was significantly diminished in the treated fistula group, although the increase in LV compliance was similar in both treated and untreated fistula hearts. MMP inhibition significantly attenuates the myocardial remodeling associated with chronic volume overload, as evidenced by prevention of dilatation, a marked reduction in LV hypertrophy, and preservation of ventricular function.

In vivo molecular target assessment of matrix metalloproteinase inhibition.

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.

Effect of matrix metalloproteinase inhibition on colonic anastomotic healing in rats

Wound strength depends on the balance between collagen synthesis and degradation; however, the role of collagen breakdown in wound healing is still not well understood. We investigated the role of matrix metalloproteinases in wound healing by using BE16627B, a matrix metalloproteinase inhibitor. Identical surgical procedures consisting of a colonic anastomosis (single-layer, inverted) and implantation of an osmotic pump in the back were performed in male Sprague-Dawley rats weighing 270 to 290 grams. The animals were randomly assigned to receive either BE16627B (n = 10) dissolved in dimethylsulfoxide and diluted with ethylene glycol at a dosage of 2.4 mg/rat/day for 3 days or the vehicle solution alone (n = 11). The solutions were administered through the surgically implanted osmotic pumps. The animals were killed 4 days after surgery, and the colonic bursting pressure (mm Hg) and hydroxyproline concentration (μg/mg wet tissue, index of collagen) were measured. The administration of BE16627B enhanced colonic anastomotic healing, as measured by the increase in the colonic bursting pressure (160 ± 12 vs. 125 ± 7 mm Hg; P <0.05) and the increase in the soluble fraction of collagen (0.27 ± 0.01 vs. 0.21 ± 0.01 μg/mg wet tissue; P < 0.01) in the anastomosis. Histologic examination of the tissue revealed that the use of BE16627B resulted in the preservation of the multilayered colonic structure and increased the network of collagen between both ends of the colon in the thickening submucosal layer. These findings demonstrate that the inhibition of matrix metalloproteinase activity influences colonic anastomotic healing, indicating a potential mechanism for enhancing anastomotic healing.

Effect of matrix metalloproteinase inhibition on pancreatic cancer invasion and metastasis: an additive strategy for cancer control.

To investigate the effect of a matrix metalloproteinase (MMP) inhibitor, BB-94, on the viability, invasion, and metastases of pancreatic cancer. Inhibitors of MMPs, enzymes that degrade extracellular matrix, have been tested as single chemotherapeutic agents for pancreatic cancer. Capan1 and AsPC1 cell lines were studied. BB-94 cytotoxicity was evaluated by cell proliferation assays. Production of MMP2 and MMP9 in conditioned media was demonstrated by gelatin zymography. The in vitro effect of BB-94 on cell invasion was assayed using invasion chambers. Hepatic metastases from pancreatic cancer were induced by intrasplenic injections of Capan1 or AsPC1 cells in nude mice. The in vivo effect of BB-94 on liver metastases was evaluated by comparing animals receiving BB-94 treatment with controls receiving vehicle alone. Variables measured included death rate and tumor burden (liver-to-body weight ratio). BB-94 was not cytotoxic between 3 and 3,000 ng/mL. Zymography demonstrated production of MMP2 and MMP9 by both cell lines, with complete inhibition of these enzymes by BB-94 at 48 ng/mL. Invasion chamber assays showed that BB-94 (48-400 ng/mL) impeded cell invasion in vitro compared with untreated controls. In vivo, BB-94 prevented death or reduced the death rate from hepatic metastases in animals injected with Capan1 or AsPC1 cells. BB-94 treatment resulted in significant reductions in hepatic tumor burden compared with untreated controls. Inhibition of MMP reduces both growth of pancreatic cancer metastases and the death rate. These actions do not reflect cytotoxicity but rather result from impaired cancer cell attachment, migration, and organ invasion. MMP inhibitors may provide an additive effect to cytotoxic agents in multidimensional treatment regimens for pancreatic cancer.

Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice.

Extracellular matrix synthesis and degradation contribute to the morphological changes that occur after myocardial infarction (MI). We tested the hypothesis that inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling in experimental MI. Seventy-one male FVB mice that survived ligation of the left anterior coronary artery were randomized to a broad-spectrum MMP inhibitor (CP-471,474) or placebo by gavage. Echocardiographic studies were performed before randomization (within 24 hours of surgery) and 4 days later and included short-axis imaging at the midpapillary and apical levels. Infarction as defined by wall motion abnormality was achieved in 79% of the procedures (n=56), and mortality rate during the 4-day protocol was 23% (9 of 36 on treatment vs 7 of 35 on placebo; P=NS). Baseline end-diastolic and end-systolic dimensions and areas were similar (P=NS) between treated and placebo groups. At follow-up, infarcted mice allocated to MMP inhibitor had significantly smaller increases in end-systolic and end-diastolic dimensions and areas at both midpapillary and apical levels compared with infarcted mice allocated to placebo (all P<0.05). In addition, infarcted animals that received MMP inhibitor had no change in fractional shortening (-3+/-13%), whereas animals that received placebo had a decrease in fractional shortening (-12+/-12%) (P<0.05). In an analysis stratified by baseline end-diastolic area, the effects of MMP inhibition on the changes in end-systolic area and end-diastolic area were most prominent in animals that had more initial left ventricular dilatation (both P<0.05). -Administration of an MMP inhibitor attenuates early left ventricular dilation after experimental MI in mice. Further studies in genetically altered mice and other models will improve understanding of the role of MMPs in left ventricular remodeling.