Transient changes in the composition of the myocardial extracellular matrix may contribute to the ventricular systolic dysfunction in Stress-induced cardiomyopathy (SIC). We examined the changes in plasma matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that occur early after the clinical presentation of SIC.
Methods and Results
Ten patients with SIC were enrolled. Plasma concentrations of the six major MMPs (MMP-1,-2,-3,-7,-8,-9) and all 4 tissue inhibitors of MMPs (TIMP-1,-2,-3,-4) were analyzed and compared with data from fifteen control subjects. Within 24 hours of the clinical presentation, SIC patients had lower MMP-1 levels (0.41±0.13 vs 0.70±0.13 pg/mL, p = 0.048) and MMP-8 levels (1.61±0.34 vs 4.84±1.38 pg/mL, p = 0.001) and higher TIMP-4 levels (3.06±0.40 vs 2.16±0.18 pg/mL, p =0.05) compared to control. Seven of nine SIC patients had elevated LV end-diastolic pressures, and all had normal LV end-diastolic dimensions and volumes.
Patients afflicted with SIC had MMPs and TIMPs profiles similar to those described in hypertensive heart disease and diastolic heart failure and different than the profiles following myocardial infarction. Our findings uncovered a unique biomolecular profile in SIC during the first 24 hours of presentation.
Metalloproteinases; Cardiomyopathy; Stress; Stress-induced cardiomyopathy
Congenital bicuspid aortic valves (BAVs) result from fusion of two valve cusps, resulting in left-noncoronary (L-N), right-left (R-L), and right-noncoronary (R-N) morphologies. BAVs predispose to ascending thoracic aortic aneurysms (ATAAs). This study hypothesized that ATAAs with each BAV morphology group possess unique signatures of matrix metalloproteinases (MMPs) and endogenous tissue inhibitors (TIMPs).
ATAA tissue from 46 BAV patients was examined for MMP/TIMP abundance and global MMP activity compared to normal aortic specimens (n=15). Proteolytic balance was calculated as the ratio of MMP abundance to a composite TIMP score (TS). Results were stratified by valve morphology group (L-N (n=6), R-L (n=31), and R-N(n=9)).
The BAV specimens (p<0.05 vs. normal aorta, 100%) displayed elevated global MMP activity (273±63%), MMP-9 (263±47%), and decreased MMP -7 (56±10%), -8 (58±11%), TIMP -1 (63±7%) and -4 (38±3%). The R-L group showed increased global MMP activity (286±89%) and MMP-9 (267±55%) with reduced MMP -7 (45±7%) -8 (68±15%), TIMP -1 (58±7%) and -4 (35±3%). The L-N group showed elevated global MMP activity (284±71%), and decreased MMP-8 (37±17%) and TIMP-4 (48±14). In the R-N group, MMP -7 (46±13%) and -8 (36±17%), and TIMP -1 (59±10) and -4 (42±5%) were decreased. The R-L group demonstrated an increased proteolytic balance for MMP-1, MMP-9, and MMP-12 relative to L-N and R-N.
Each BAV morphology group possesses a unique signature of MMPs and TIMPs. MMP/TIMP score ratios suggest that the R-L group may be more aggressive, justifying earlier surgical intervention.
MicroRNAs (miRs) are small noncoding RNAs that recognize and bind to mRNAs and inhibit protein translation or degrade mRNA. Studies in animal models have suggested that miRs play a translational or post-translational regulatory role in myocardial growth, fibrosis, viability, and remodeling. However, whether specific temporal changes in miRs occur in patients during the LV remodeling process that follows a myocardial infarction (post-MI) remains unknown. The purpose of the current pilot study was to test the hypothesis that plasma miRs could be reliably measured in post-MI patients and that there is the relationship between temporal changes in specific miRs and post-MI LV structural remodeling.
Methods and Results
LV end-diastolic volume (EDV, echocardiography) and plasma miR were measured in age matched referent controls (CTL n=12) and post-MI patients (n=12) from day 2 through day 90 post-MI. Selected miRs (miR-1, -21, -29a, 133a, 208) were measured using quantitative rt-PCR and normalized for endogenous snRNA U6. Following MI, LVEDV increased progressively compared to CTL; this was accompanied by time dependent changes in specific miRs. For example, miR-21 initially fell 2 days post-MI (0.3±0.1 fold vs. CTL, p< 0.05), increased 5 days post-MI (2±1 fold vs. CTL, p< 0.05), and returned to CTL values at later post-MI time points. In contrast, miR-29a increased 5 days post-MI (4±1 fold vs. CTL, p< 0.05) and then fell to CTL at later time points. miR-208 increased 5 days post-MI (3±1 fold vs. CTL, p< 0.05) and remained elevated up to 90 days post-MI.
A time-dependent change in miRs occurred in post-MI patients that included an early and robust rise in miRs that have been shown to affect myocardial growth, fibrosis and viability. Thus, serially profiling miRs in the plasma of post-MI patients may hold both mechanistic and prognostic significance.
Myocardial Infarction; Remodeling; microRNA
Patients with severe left ventricular (LV) pressure overload (LVPO) secondary to aortic stenosis can present with signs and symptoms of heart failure despite normal LV ejection fractions (LVEF). This process occurs, at least in part, as a result of LVPO-induced extracellular matrix (ECM) remodeling which promulgates increased LV stiffness and impaired diastolic function. However, the determinants which drive ECM remodeling in this form of LVPO remain to be fully defined.
LVPO was induced in mature pigs (n=15) by progressive ascending aortic cuff inflation (once/week/4 weeks) whereby LV mass, LVEF, and regional myocardial stiffness (rKm) were compared to referent controls (n=12). Determinants of ECM remodeling were assessed by measuring levels of mRNA expression for fibrillar collagens, matrix metalloproteinases (MMPs), and the tissue inhibitors of MMP-1 and -4 (TIMP-1, -4).
With LVPO, LV mass and rKm increased by 2- and 3-fold, respectively, compared to control, with no change in LVEF. LV myocardial collagen increased approximately 2-fold which was accompanied by reduced solubility (i.e. increased cross-linking) with LVPO, but mRNA expression for fibrillar collagen and MMPs remained relatively unchanged. In contrast, a robust increase in mRNA expression for TIMP-1 and -4 occurred with LVPO.
In a progressive model of LVPO, which recapitulates the phenotype of aortic stenosis, increased ECM accumulation and subsequently increased myocardial stiffness was not due to increased fibrillar collagen expression, but rather due to determinants of post-translational control which included increased collagen stability (thereby resistant to MMP degradation) and increased endogenous MMP inhibition. Targeting these ECM post-translational events with LVPO may hold both diagnostic and therapeutic relevance.
Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. While alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored.
Methods and Results
The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared to normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Using a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens.
These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs, and may provide significant targets for therapeutic and diagnostic applications.
aneurysm; thoracic aorta; microRNA; MMP; remodeling
While localized delivery of biocomposite materials, such as calcium hydroxyapatite (CHAM), have been demonstrated to potentially attenuate adverse LV remodeling post-myocardial infarction (MI), the underlying biological mechanisms for this effect remain unclear. This study tested the hypothesis that targeted CHAM injections would alter proteolytic pathways (matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs)), and be associated with parameters of post-MI LV remodeling.
Methods and Results
MI was induced in adult sheep followed by 20 targeted injections of a total volume of 1.3 mL (n=6) or 2.6 mL of CHAM (n=5), or saline (n=13), and LV end-diastolic volume (EDV) and MMP/TIMP profiles in the MI region were measured at 8 weeks post-MI. LV EDV decreased with 2.6 mL CHAM vs MI Only (105.4±7.5 vs 80.6±4.2 respectively, p<0.05) but not with 1.3 mL CHAM (94.5±5.0, p=0.32). However, MI thickness increased by 2-fold in both CHAM groups compared to MI Only (p<0.05). MMP-13 increased 40-fold in the MI Only group (p<0.05) but fell by over 6-fold in both CHAM groups (p<0.05). MMP-7 increased approximately 1.5-fold in the MI Only group (p<0.05) but decreased to referent control values in both CHAM groups in the MI region (p<0.05). Collagen content was reduced by approximately 30% in the CHAM groups compared to MI Only (p<0.05).
Differential effects on LV remodeling and MMP/TIMP profiles occurred with CHAM. Thus, targeted injections of a biocomposite material can favorably affect the post-MI remodeling process and therefore holds promise as a treatment strategy in and of itself, or as a matrix with potentially synergistic effects with localized pharmacologic or cellular therapies.
Infarct Expansion; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are known to modulate left ventricular (LV) remodeling after a myocardial infarction (MI). However, the temporal and spatial variation of MMP activation and their relationship to mechanical dysfunction post MI remains undefined.
Methods and Results
MI was surgically induced in pigs (n=23) and cine MR and dual isotope hybrid SPECT/CT imaging obtained using thallium-201 (201Tl) and a technetium-99m labeled MMP targeted tracer (99mTc-RP805) at 1, 2 and 4 weeks post MI along with controls (n=5). Regional myocardial strain was computed from MR images and related to MMP zymography and ex vivo myocardial 99mTc-RP805 retention. MMP activation as assessed by in vivo and ex vivo 99mTc-RP805 imaging/retention studies was increased nearly 5-fold within the infarct region at 1 week post-MI and remained elevated up to 1 month post-MI. The post-MI change in LV end-diastolic volumes was correlated with MMP activity (y=31.34e0.48x, p=0.04). MMP activity was increased within the border and remote regions early post-MI, but declined over 1 month. There was a high concordance between regional 99mTc-RP805 uptake and ex vivo MMP-2 activity.
A novel, multimodality non-invasive hybrid SPECT/CT imaging approach was validated and applied for in vivo evaluation of MMP activation in combination with cine MR analysis of LV deformation. Increased 99mTc-RP805 retention was seen throughout the heart early post-MI and was not purely a reciprocal of 201Tl perfusion. 99mTc-RP805 SPECT/CT imaging may provide unique information regarding regional myocardial MMP activation and predict late post-MI LV remodeling.
MMP; LV remodeling; spatiotemporal imaging
Antifibrinolytic therapy, such as the use of the serine protease inhibitor aprotinin, was a mainstay for hemostasis following cardiac surgery. However, aprotinin was empirically dosed, and while the pharmacological target was the inhibition of plasmin activity (PLact) this was never monitored, off-target effects occurred, and led to withdrawn from clinical use. The present study developed a validated fluorogenic-microdialysis method to continuously measure PLact and tested the hypothesis that standardized clinical empirical aprotinin dosing would impart differential and regional effects on PLact.
Pigs (30 kg) were instrumented with microdialysis probes to continuously measure PLact in myocardial, kidney and skeletal muscle compartments (deltoid) and then randomized to High Dose aprotinin administration (2 mKIU load/ 0.5 mKIU/hr infusion; n=7), Low Dose aprotinin administration (1 mKIU load/ 0.250 mKIU/hr infusion; n=6). PLact was compared to time matched vehicle (n=4), and PLact was also measured in plasma by an in-vitro fluorogenic method. Aprotinin suppressed PLact in the myocardium and kidney at both High and Low Doses- indicative that both doses exceeded a minimal concentration necessary for PLact inhibition. However, differential effects of aprotinin on PLact were observed in the skeletal muscle indicative of different compartmentalization of aprotinin.
Using a large animal model and a continuous method to monitor regional plasmin activity, these unique results demonstrated that an empirical aprotinin dosing protocol causes maximal and rapid suppression in the myocardium and kidney and in turn would likely increase the probability of off-target effects and adverse events. Further, this proof of principle study demonstrated that continuous monitoring of determinants of fibrinolysis may provide a novel approach for managing fibrinolytic therapy.
aprotinin; plasmin inhibition; fibrinolysis; plasma; interstitial; microdialysis probe
The left ventricular phenotype of idiopathic dilated cardiomyopathy (DCM) can appear similar in paediatric and adult patients. However, the aetiology of paediatric DCM is usually idiopathic, and often leads an aggressive clinical course. A structural underpinning of DCM is extracellular matrix changes, which are determined by a balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). This study tested the hypothesis that different MMP/TIMP profiles occur in paediatric and adult DCM patients.
Methods and results
Left ventricular samples from paediatric (age 9 ± 5 years; n = 10) and adult (age 62 ± 3 years; n = 20) DCM (at time of transplant) were subjected to an MMP/TIMP multiplex array and immunoassay in order to measure the MMP subclasses; collagenases (MMP-8, -13), gelatinases (MMP-2, -9), stromelysin/matrilysin (MMP-3, -7), membrane type (MT1-MMP), as well as for the four known TIMPs. MMP-8 and -9 levels increased by over 150% (P < 0.05), whereas MMP-3 and -7 levels decreased by over 30% (P < 0.05) in paediatric DCM when compared with adult DCM. TIMP-1 and -2 levels increased two-fold (P < 0.05), but TIMP-3 fell by 41% (P < 0.05) in paediatric DCM. Myocardial levels of specific interleukins (IL-1beta, IL-2, IL-8) were increased by approximately 50% in paediatric DCM.
These unique findings demonstrated that a specific MMP/TIMP profile occurs in paediatric DCM when compared with adult DCM, and that local cytokine induction may contribute to this process. These distinct differences in the determinants of myocardial matrix structure and function may contribute to the natural history of DCM in children.
Cardiomyopathy; Extracellular matrix; Matrix metalloproteinase; Paediatrics
Congenital heart surgery initiates a complex inflammatory response that can influence the post-operative course. However, broad integration of the cytokine and proteolytic cascades (matrix metalloproteinases: MMPs), which may contribute to post-operative outcomes has not been performed.
Using a low volume (50 – 60 μL), high sensitivity, multiplex approach a panel of cytokines (IL-2, -4, -6, -8, -10, TNFα, IL-1β, GM-CSF) and MMPs (MMP-2, -3, -7, -8, -9, -12, -13) were serially measured in patients (n=9) pre-operatively and post-VSD repair. Results were correlated with outcomes such as inotropic requirement, oxygenation, and fluid balance. Serial changes in peri-operative plasma levels of the cytokines and MMPs exhibited distinct temporal profiles. Plasma levels of IL-2, -8, -10, and MMP-9 peaked within 4 hours, while MMP-3 and MMP-8 levels remained elevated at 24 and 48 hours following cross-clamp removal. Area-under-the-curve analysis of early cytokine levels were associated with major clinical variables, including inverse correlations between early IL-10 levels and cumulative inotrope requirement at 48 hours (r: −0.85, p<0.005) and late MMP-7 levels and cumulative fluid balance (r: −0.90, p<0.001).
The unique findings of this study were that serial profiling a large array of cytokines and proteolytic enzymes following congenital heart surgery can provide insight into relationships between changes in bioactive molecules to early postoperative outcomes. Specific patterns of cytokine and MMP release may hold significance as biomarkers for predicting and managing the post-operative course following congenital heart surgery.
Ventricular septal defect repair; Inflammation; Cytokines; Matrix metalloproteinases
Thoracic aortic aneurysms (TAAs) result from dysregulated remodeling of the vascular extracellular matrix (ECM) which may occur as a result of altered resident cellular function. The present study tested the hypothesis that aortic fibroblasts undergo a stable change in cellular phenotype during TAA formation.
Primary murine aortic fibroblasts were isolated from normal and TAA-induced aortas (4-wks post-induction with 0.5M CaCl2 15 min) by outgrowth method. Normal and TAA cultures were examined using a focused PCR array to determine fibroblast-specific changes in gene expression in the absence and presence of biological stimulation (endothelin-1, phorbol-12-myristate-13-acetate, angiotensin II). The relative expression of 38 genes, normalized to 4 housekeeping genes, was determined and genes displaying a minimum 2-fold increase/decrease or genes with significantly different normalized Ct values were considered to have altered expression.
At steady state TAA fibroblasts revealed elevated expression of several MMPs (Mmp2, Mmp11, Mmp14), collagen genes/elastin (Col1a1, Col1a2, Col3a1, Eln), and other matrix proteins, as well as decreased expression of Mmp3, Timp3, and Ltbp1. Moreover, gene expression profiles in TAA fibroblasts were different than normal fibroblasts after equivalent biological stimuli.
This study demonstrated for the first time that isolated primary aortic fibroblasts from TAA-induced mice possess a unique and stable gene expression profile, and when challenged with biological stimuli, induce a transcriptional response that is different from normal aortic fibroblasts. Together, these data suggest that aortic fibroblasts undergo a stable phenotypic change during TAA development which may drive the enhancement of ECM proteolysis in TAA progression.
aneurysm; extracellular matrix; remodeling; phenotype; fibroblast
Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contribute to left ventricular (LV) wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high frequency stimulation (LHFS) using low amplitude electrical pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning.
At 3 wks following MI, pigs were randomized for LHFS (n=7, 240bpm, 0.8V, 0.05ms pulses) or unstimulated (UNSTIM, n=10). At 4 wks post-MI, LV wall thickness (echo, 0.89±0.07 vs 0.67±0.08 cm, p<0.05) and regional stiffness (piezoelectric crystals, 14.70±2.08 vs 9.11±1.24, p<0.05) were higher with LHFS than UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage, 943±59 vs 1210±72 units, p<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower, while TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all p<0.05). Transforming growth factor-β (TGF-β) receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electrical stimulation (4Hz) of isolated fibroblasts resulted in a reduction of MMP-2 and MT1-MMP levels, but increased TIMP-1 levels compared to unstimulated fibroblasts.
These unique findings demonstrate that LHFS of the MI region altered LV wall thickness and material properties, likely due to reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify LV remodeling post-MI.
Myocardial infarction; extracellular matrix; remodeling; matrix metalloproteinases
Following cardiopulmonary bypass (CPB), elaboration of cytokines, and subsequent interstitial proteases induction, such as matrix metalloproteinases (MMPs), can result in a complex postoperative course. The serine protease inhibitor, aprotinin, which has been used in congenital heart surgery putatively for modulating fibrinolysis, is now unavailable, necessitating the use of lysine analogues, such as tranexamic acid (TXA). The present study tested the hypothesis that distinctly different plasma profiles of signaling molecules and proteases, would be differentially affected following the administration of aprotinin or TXA in the context of congenital cardiac surgery and CPB.
37 Patients (age 4.8±0.3 mos) undergoing corrective surgery for ventricular septal defect (VSD) and Tetralogy of Fallot (TOF) received either aprotinin (n=22), or TXA (n=15). Using a high throughput multiplex suspension immunoassay, plasma was serially quantified for cytokines and MMPs: before aprotinin or TXA (baseline), after separation from CPB, and 4, 12, 24, and 48 hours post-CPB.
Tumor necrosis factor-alpha (TNF) increased initially following CPB in both the aprotinin and TXA groups, but at 24 and 48 hours post-CPB was approximately 50% lower in the aprotinin group (p<0.05). IIL-10 levels were 3-fold higher in the TXA group compared to the aprotinin group immediately post-CBP (p<0.05). Plasma levels of MMP types associated with inflammation, MMP-8 and -9, were 2-fold higher in the late post-CPB period in the TXA group when compared to the aprotinin group.
Following VSD or TOF repair in children, cytokine induction occurs, which is temporally related to the emergence of a specific MMP profile. Moreover, these unique findings demonstrated differential effects between the serine protease inhibitor aprotinin, and the lysine analogue TXA with respect to cytokine and MMP induction in the early post-operative period. The different cytokine-proteolytic profile between these anti-fibrinolytics may in turn influence biological processes in the post-operative period.
Vascular remodeling relies upon extracellular matrix restructuring by the matrix metalloproteinases (MMPs). Induction of MMP-2 and MMP-9 by biological signaling molecules has been defined, but whether a mechanical stimulus such as elevated wall tension may generate MMP promoter activation remains unknown. Accordingly, this study examined whether MMP promoter activation would occur as a function of wall tension.
Materials and Methods
The MMP-2 or MMP-9 promoter sequences were fused to the reporter gene lacZ and inserted into the mouse genome. Thoracic aortic rings were harvested (6 preparations/construct) and maintained under physiological conditions at predetermined tension values corresponding to 0, 70, 85 and 100 mmHg for 3 hours. Relative gene expression of lacZ, directly reflecting MMP promoter activity, was then quantified by QPCR.
MMP-2 promoter activity decreased to 0.42±0.11 at 0 mmHg and increased to 1.57±0.24 fold at 100 mmHg (p<0.05), whereas MMP-9 was unaffected.
Using unique transgenic constructs with homology to human MMP promoters, this study demonstrated that a physiologically relevant mechanical stimulus was sufficient to differentially induce MMP promoter activation.
vascular remodeling; wall tension; thoracic aorta; matrix metalloproteinase
Dilated cardiomyopathy is an important cause of heart failure in both children and adults, but is more progressive in children. In adult dilated cardiomyopathy, left ventricular remodeling is associated with changes in plasma levels of matrix metalloproteinases, and tissue inhibitor of metalloproteinases. Plasma matrix metalloproteinases, and tissue inhibitors of metalloproteinase changes in pediatric dilated cardiomyopathy has not been examined. The present study developed a low blood volume, high sensitivity assay to test the hypothesis that unique and differential plasma matrix metalloproteinases and tissue inhibitors of metalloproteinase profile exists in pediatric dilated cardiomyopathy patients.
A systemic blood sample (1 milliliter) was obtained from 7 children with dilated cardiomyopathy (age 8 plus or minus 7 years), and 26 age-matched normal volunteers. Using a high throughput multiplex suspension immunoassay, plasma levels were quantified for: collagenases (matrix metalloproteinase-8), gelatinases (matrix metalloproteinase-2, -9), lysins (matrix metalloproteinase-3, -7), and tissue inhibitor of metalloproteinases-1, -2, and -4. Matrix metalloproteinase to tissue inhibitor of metalloproteinases ratios were also calculated. Plasma matrix metalloproteinase-2, -7, -8 and -9 levels were increased by greater than 2-fold in dilated cardiomyopathy patients than normals ( with p less than 0.05). Dilated cardiomyopathy patients also had significantly higher tissue inhibitor of metalloproteinases-1 and -4 (298 percent and 230 percent; with p less than 0.05).
These unique findings demonstrated that a specific plasma matrix metalloproteinase/tissue inhibitors of metalloproteinase profile occurs in pediatric dilated cardiomyopathy when compared to normal children. These distinct differences in the determinants of myocardial matrix structure and function may contribute to the natural history of dilated cardiomyopathy in children, and may provide a novel biomarker platform in pediatric dilated cardiomyopathy.
The matrix metalloproteinases (MMPs) have been implicated in the aggressive course of non-small cell lung cancer (NSCLC). However, there are a large number of MMP subtypes with diverse proteolytic substrates and different induction pathways. This study tested the hypothesis that a differential MMP profile would exist between NSCLC and normal lung and that MMP patterns would differ between NSCLC histologic type.
NSCLC samples and remote normal samples were obtained from patients with stage I or II NSCLC with either squamous cell (n=22) or adenocarcinoma (n=19) histology. Absolute concentrations for each of the MMP subclasses; collagenases (MMP-1, 8, -13), gelatinases (MMP-2,-9), lysins (MMP-2, -7) and elastase (MMP-12) were determined by a calibrated and validated multiplex suspension array.
Overall, MMP levels were significantly increased in NSCLC compared to normal. For example, MMP-1 and MMP-7 increased by approximately 10 fold in NSCLC (p<0.05). Moreover, a different MMP portfolio was observed between NSCLC histologic types. For example MMP-1,-8,-9 and -12 increased by over 4-fold in squamous cell versus adenocarcinoma (p<0.05). In those patients who recurred within 3 years of resection, 3-fold higher levels of MMP-8 and -9 were observed (p<0.05).
Increased levels of a number of MMP types occur with NSCLC, but the MMP profile was distinctly different between histologic types and in those patients with recurrence. These different MMP profiles may be important in the mechanistic basis for the natural history of different NSCLC types, as well as identifying potential prognostic and therapeutic targets.
matrix metalloproteinases; lung cancer; multiplex; recurrence
Myocyte death occurs by necrosis and caspase-mediated apoptosis in myocardial infarction (MI). In-vitro studies suggest caspase activation causes myocardial contractile protein degradation without inducing apoptosis. Thus, caspase activation may evoke left ventricular (LV) remodeling through independent processes post-MI. The effects of caspase activation on LV geometry post-MI remain unclear. This project applied pharmacologic caspase inhibition (CASPI) to a porcine model of MI.
Methods and Results
Pigs (34kg) were instrumented in order to induce 60min of coronary artery occlusion followed by reperfusion and a 7-day follow-up period. Upon reperfusion, the pigs were randomized to saline (n=12) or CASPI (n=10, IDN6734, 6mg/kg IV, then 6mg/kg/hr for 24hrs). Plasma troponin-I values were reduced with CASPI compared to saline at 24hrs post-MI (133±15 vs 189±20ng/mL, respectively, p<0.05). LV end-diastolic area (echocardiography) and inter-regional length (sonomicrometry) increased from baseline in both groups but were attenuated with CASPI by 40% and 90%, respectively (p<0.05). Myocyte length was reduced with CASPI compared to saline (128±3 vs 141±4μm, respectively, p<0.05). Plasma free-pro-MMP-2 values increased from baseline with CASPI (27±6%, p<0.05) indicative of reduced conversion to active MMP-2. Separate in-vitro studies demonstrated that activated caspase species cleaved pro-MMP-2 yielding active MMP-2 forms and that MMP activity was increased in the presence of activated caspase-3.
CASPI attenuated regional and global LV remodeling post-MI and altered viable myocyte geometry. Caspases increased MMP activity in-vitro while CASPI modified conversion of MMP-2 to the active form in-vivo. Taken together, the results of the present study suggest that the elaboration of caspases post-MI likely contribute to LV remodeling through both cellular and extracellular mechanisms.
myocardial infarction; remodeling; caspase inhibition; left ventricular
Targeted delivery of mesenchymal precursor cells (MPCs) can modify left ventricular (LV) cellular and extracellular remodeling following myocardial infarction (MI). However, whether and to what degree LV remodeling may be affected by MPC injection post-MI, and whether these effects are concentration dependent remain unknown.
Methods and Results
Allogeneic MPCs were expanded from sheep bone marrow, and direct intra-myocardial injection was performed within the borderzone region one hour following MI induction (coronary ligation) in sheep at the following concentrations: 25×106 (25 M, n=7), 75 ×106 (75 M, n=7), 225 ×106 (225 M, n=10), 450 ×106 (450 M, n=8), and MPC free media only (MI Only, n=14). LV end diastolic volume increased in all groups but was attenuated in the 25 and 75 M groups. Collagen content within the borderzone region was increased in the MI Only, 225, and 450 M groups. Whereas plasma ICTP, an index of collagen degradation, was highest in the 25 M group. Within the borderzone region matrix metalloproteinases (MMPs) and MMP tissue inhibitors (TIMPs) also changed in a MPC concentration dependent manner. For example, borderzone levels of MMP-9 were highest in the 25 M group when compared to the MI Only and other MPC treatment group values.
MPC injection altered collagen dynamics, MMP, and TIMP levels in a concentration dependent manner, and thereby influenced indices of post-MI LV remodeling. However, the greatest effects with respect to post-MI remodeling were identified at lower MPC concentrations, thus suggesting a therapeutic threshold exists for this particular cell therapy.
Infarct Expansion; Stem Cells; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinase; Fibrosis
The effect of multiple integrated stimuli on vascular wall expression of matrix metalloproteinases (MMPs) remains unknown. Accordingly, this study has examined the influence of the vasoactive peptide angiotensin II (AngII) on wall tension-induced promoter activation of MMP-2, MMP-9, and membrane type-1 MMP (MT1-MMP).
Methods and Results
Thoracic aortic rings harvested from transgenic reporter mice containing the MMP-2, MMP-9, or MT1-MMP promoter sequence fused to a reporter gene were subjected to three hours of wall tension at 70, 85, or 100 mmHg with or without 100nM AngII. Total RNA was harvested from the aortic rings, and reporter gene transcripts were quantified by QPCR to measure MMP promoter activity. MT1-MMP promoter activity was increased at both 85 and 100 mmHg compared to baseline tension of 70 mmHg, while treatment with AngII stimulated MT1-MMP promoter activity to the same degree at all tension levels (p<0.05). Elevated tension and AngII displayed a potential synergistic enhancement of MMP-2 promoter activation at 85 and 100mmHg, while the same stimuli caused a decrease in MMP-9 promoter activity (p<0.05) at 100 mmHg.
This study has demonstrated that exposure to a relevant biological stimulus (AngII) in the presence of elevated tension modulated MMP promoter activation. Furthermore, these data suggest that a mechanical-molecular set point exists for the induction of MMP promoter activation, and that this set point can be adjusted up or down by a secondary biological stimulus. Together, these results may have significant clinical implications toward the regulation of hypertensive vascular remodeling.
Metalloproteinases; Aorta; Wall stress; Angiotensin
The direct consequences of a persistently increased myocardial expression of the unique matrix metalloproteinase (MMP), membrane type-1 (MT1-MMP) on myocardial remodeling remained unexplored.
Cardiac restricted MT1-MMPexp was constructed in mice using the full length human MT1-MMP gene ligated to the myosin heavy chain promoter, which yielded approximately a 200% increase in MT1-MMP when compared to age/strain matched wild type mice (WT). LV function and geometry was assessed by echocardiography in 3 month (“young”) WT (n=32) and MT1-MMPexp (n=20) mice, and compared to 14 month (“middle age”) WT (n=58) and MT1-MMPexp (n=35) mice. LV end-diastolic volume was similar between the WT and MT1-MMPexp young groups as was LV ejection fraction. In the middle age WT mice, LV end-diastolic volume and ejection fraction was similar to young WT mice. However, in the MT1-MMPexp middle age mice, LV end-diastolic volume was approximately 43% higher and LV ejection fraction 40% lower (both p<0.05). Moreover, in the middle age MT1-MMPexp mice, myocardial fibrillar collagen increased by nearly 2-fold and was associated with an approximate 3-fold increase in the processing of the pro-fibrotic molecule, latency-associated transforming growth factor binding protein. In a second study, 14 day survival following myocardial infarction was significantly lower in middle aged MT1-MMPexp mice.
Persistently increased myocardial MT1-MMP expression, in and of itself, caused LV remodeling, myocardial fibrosis, dysfunction and reduced survival following myocardial injury. These findings suggest that MT1-MMP plays a mechanistic role in adverse remodeling within the myocardium.
matrix; myocardial remodeling; ventricular function; aging
Epsilon aminocaproic acid (EACA) is used in cardiac surgery to modulate plasmin activity (PLact). The present study developed a fluorogenic-microdialysis system to measure in-vivo region specific temporal changes in PLact following EACA administration.
Pigs (25-35kg) received EACA (75mg/kg, n=7) or saline in which microdialysis probes were placed in the liver, myocardium, kidney and quadricep muscle. The microdialysate contained a plasmin specific fluorogenic peptide and fluorescence emission, which directly reflected PLact, determined at baseline, 30, 60, 90 and 120 minutes following EACA/vehicle infusion.
EACA caused significant decreases in liver and quadricep PLact at 60, 90, 120 minutes and at 30, 60, 120 minutes respectively (p<0.05). In contrast, EACA induced significant biphasic changes in heart and kidney PLact profiles with initial increases followed by decreases at 90 and 120 minutes (p<0.05). The peak EACA interstitial concentrations for all compartments occurred at 30 minutes post infusion, and were 5-fold higher in the renal compartment and 4-fold higher in the myocardium, when compared to the liver or muscle (p<0.05).
Using a large animal model and in-vivo microdialysis measurements of plasmin activity, the unique findings from this study were 2-fold. First, EACA induced temporally distinct plasmin activity profiles within the plasma and interstitial compartments. Second, EACA caused region specific changes in plasmin activity profiles. These temporal and regional heterogeneic effects of EACA may have important therapeutic considerations when managing fibrinolysis in the perioperative period.
A major complication associated with cardiac surgery is excessive and prolonged bleeding in the perioperative period. Improving coagulation by inhibiting fibrinolysis, primarily through inhibition of plasmin activity (PLact) with antifibrinolytics such as tranexamic acid (TXA) has been a pharmacological mainstay in cardiac surgical patients. Despite its almost ubiquitous use, the temporal and regional modulation of PLact profiles by TXA remain unexplored. Accordingly, the present study developed a fluorogenic-microdialysis system to measure in-vivo dynamic changes in PLact following TXA administration in a large animal model.
Pigs (25–35kg) were randomly assigned to receive TXA (30mg/kg, diluted into 50cc normal saline; n=9) or vehicle (50cc normal saline; n=7) in which microdialysis probes were placed in the liver, myocardium, kidney and quadricep muscle compartments. The microdialysate infusion contained a validated plasmin specific fluorogenic peptide. The fluorescence emission (Standard Fluorogenic Units; SFU) of the interstitial fluid collected from the microdialysis probes, which directly reflects PLact, was determined at steady-state baseline, 30, 60, 90 and 120 minutes following TXA/vehicle infusion. Plasma PLact was determined at the same time points using the same fluorogenic substrate approach.
With respect to vehicle values, TXA reduced plasma PLact at 30 minutes post infusion by more than 110 SFU (p<0.05). Specifically, there was a decrease in liver PLact at 90 and 120 minutes post TXA infusion of greater than 150 (p<0.05) and 175 (p<0.05) SFU, respectively. The decline in liver PLact occurred 60 minutes after the maximal decline in plasma PLact. In contrast, kidney, heart, and quadriceps PLact transiently increased followed by an overall decrease at 120 minutes.
Using a large animal model and in-vivo microdialysis measurements of PLact, the unique findings from this study were 2-fold. First, TXA induced temporally distinct PLact profiles within the plasma and selected interstitial compartments. Second, TXA caused region specific changes in PLact profiles. These temporal and regional differences in the effects of TXA may have important therapeutic considerations when managing fibrinolysis in the perioperative period.
tranexamic acid; microdialysis; interstitial plasmin; regional activity
Periods of ischemia-reperfusion (I/R) during cardiac surgery are associated with transient left ventricular (LV) dysfunction and an inflammatory response. The goal of this study was to examine potential dose-dependent effects of aprotinin (APRO) on LV contractility and cytokine release in the setting of I/R.
An index of LV contractility, LV maximal elastance (Emax), was measured at baseline, 30 min of ischemia, and 60 min of reperfusion by microtransducer volumetry. Mice were randomized as follows: a) APRO 20,000 kallikrein-inhibiting units (KIU)/kg (n=11); b) APRO 4×104 KIU/kg (n=10); c) APRO 8 ×104 KIU/kg (n=10) and d) Vehicle (saline; n=10). APRO doses were calculated to reflect half, full, and twice the clinical Hammersmith dosing schedule. Following I/R, plasma was collected for cytokine measurements.
Following I/R, Emax decreased from the baseline value by over 40% in the Vehicle group as well as in the APRO 4 ×104 KIU/kg and APRO 8 ×104 KIU/kg groups (p<0.05). However, Emax returned to near baseline values in the APRO 2 ×104 KIU/kg group. TNF increased ten-fold following I/R, but it was reduced with higher APRO doses.
This study demonstrated that a low dose of APRO provided protective effects on LV contractility, whereas higher doses suppressed TNF release. These unique findings suggest that there are distinct and independent mechanisms of action of APRO in the context of I/R.
aprotinin; ischemia-reperfusion; LV systolic elastance; cytokines; contractility
Basic studies have suggested that cross-talk exists between the endothelin-A receptor (ET-AR) and tumor necrosis factor signaling pathway. This study tested the hypothesis that administration of an ET-AR antagonist at the separation from cardiopulmonary bypass would alter the tumor necrosis factor activation in the early post operative period.
Patients (n = 44) were randomized to receive bolus infusion of vehicle, 0.1, 0.5, 1, or 2 mg/kg of the ET-AR antagonist (sitaxsentan) at the separation from cardiopulmonary bypass (n=9, 9, 9, 9, and 8 respectively). Plasma levels of tumor necrosis factor-α and soluble tumor necrosis factor receptor 1 and 2 were measured.
Compared to the vehicle group at 24 hours, plasma levels of tumor necrosis factor-α and tumor necrosis factor receptor 2 (indicative of receptor activation) were reduced in the 1 mg/kg ET-AR antagonist group (by ~13 pg/mL and ~0.5 ng/mL respectively; p<0.05). Plasma tumor necrosis factor receptor I levels also decreased (by ~1 ng/mL) following infusion of the higher doses of the ET-AR antagonist and remained lower (by ~3 ng/mL) at 24 hours post infusion (p<0.05). In addition, a dose effect was observed between the ET-AR antagonist and these indices of tumor necrosis factor activation (p<0.01).
This study demonstrated a mechanistic relationship between the ET-AR and tumor necrosis factor receptor activation in the post cardiac surgery period. Thus, in addition to the potential cardiovascular effects, a selective ET-AR antagonist can modify other biological processes relevant to the post cardiac surgery setting. (word count:245/250)
Cardiopulmonary Bypass (CPB); Inflammation; Cytokines
Left ventricular (LV) remodeling after myocardial infarction (MI) commonly causes infarct expansion (IE). This study sought to interrupt IE through microinjections of a biocompatible composite material into the post-MI myocardium.
MI was created in 21 pigs (coronary ligation). Radiopaque markers (2-mm diameter) were placed for IE (fluoroscopy). Pigs were randomized for microinjections (25 injections; 2- × 2-cm array; 200 µL/injection) at 7 days post-MI of a fibrin-alginate composite (Fib-Alg; fibrinogen, fibronectin, factor XIII, gelatin-grafted alginate, thrombin; n = 11) or saline (n = 10).
At 7 days after injection (14 days post-MI), LV posterior wall thickness was higher in the Fib-Alg group than in the saline group (1.07 ± 0.11 vs 0.69 ± 0.07 cm, respectively, p = 0.002). At 28 days post-MI, the area within the markers (IE) increased from baseline (1 cm2) in the saline (1.71 ± 0.13 cm2, p = 0.010) and Fib-Alg groups (1.44 ± 0.23 cm2, p < 0.001). However, the change in IE at 21 and 28 days post-MI was reduced in the Fib-Alg group (p=0.043 and p=0.019). Total collagen content within the MI region was similar in the saline and Fib-Alg groups (12.8 ± 1.7 and 11.6 ± 1.5 µg/mg, respectively, p = NS). However, extractable collagen, indicative of solubility, was lower in the Fib-Alg group than the saline group (59.1 ± 3.5 vs 71.0 ± 6.1 µg/mL, p = 0.020).
Targeted myocardial microinjection of the biocomposite attenuated the post-MI decrease in LV wall thickness and infarct expansion. Thus, intraoperative microinjections of biocompatible material may provide a novel approach for interrupting post-MI LV remodeling.