Christina B. Logdon

Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition

Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodeling. While MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application due to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP over-expression following a myocardial infarction (MI) significantly reduced MMP activity and attenuated adverse left ventricular remodeling in a porcine model of MI. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.

Local Hydrogel Release of Recombinant TIMP-3 Attenuates Adverse Left Ventricular Remodeling After Experimental Myocardial Infarction

Delivery of a hydrogel providing sustained release of recombinant TIMP-3 attenuated adverse ventricular remodeling after myocardial infarction in pigs. Hydrogel-Inhibitor Combo Stops Heart Damage After a heart attack, or myocardial infarction (MI), the heart tries to repair itself. This natural process is well intentioned but results in infarct expansion, scar formation, and, in turn, reduced heart function. To prevent such adverse remodeling, Eckhouse and colleagues designed an injectable hydrogel that inhibits the activity of enzymes directly involved in tissue repair. Matrix metalloproteinases (MMPs) are enzymes that are activated in heart tissue after MI. The authors encapsulated TIMP-3 (tissue inhibitor of metalloproteinase 3) in hyaluronic acid hydrogels. The gel/TIMP-3 combo or a control gel without the inhibitor was injected into the hearts of pigs after a heart attack. Weeks later, heart function, inflammation, and remodeling were evaluated. Animals administered the hydrogel with TIMP-3 had improved heart function [as determined by the left ventricular ejection fraction (LVEF)], improved LV geometry, and reduced infarct size. This local delivery mechanism could be used in the context of surgery, such as during coronary revascularization after a heart attack. Because it has been tested in pigs—which have similar heart anatomy to humans—and because other hydrogels, like alginate, have been tested in the human heart before, it is possible that this gel-inhibitor combination therapy could advance to clinical trials in the near future. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to the left ventricle (LV) remodeling that occurs after myocardial infarction (MI). However, translation of these observations into a clinically relevant, therapeutic strategy remains to be established. The present study investigated targeted TIMP augmentation through regional injection of a degradable hyaluronic acid hydrogel containing recombinant TIMP-3 (rTIMP-3) in a large animal model. MI was induced in pigs by coronary ligation. Animals were then randomized to receive targeted hydrogel/rTIMP-3, hydrogel alone, or saline injection and followed for 14 days. Instrumented pigs with no MI induction served as referent controls. Multimodal imaging (fluoroscopy/echocardiography/magnetic resonance imaging) revealed that LV ejection fraction was improved, LV dilation was reduced, and MI expansion was attenuated in the animals treated with rTIMP-3 compared to all other controls. A marked reduction in proinflammatory cytokines and increased smooth muscle actin content indicative of myofibroblast proliferation occurred in the MI region with hydrogel/rTIMP-3 injections. These results provide the first proof of concept that regional sustained delivery of an MMP inhibitor can effectively interrupt adverse post-MI remodeling.

Cyclosporin A in left ventricular remodeling after myocardial infarction

Recent studies suggest that an increase in apoptosis within the myocardium may be a contributing factor for the progression of late adverse left ventricular (LV) remodeling following myocardial infarction (MI). Given that apoptosis is often triggered by induction of the mitochondrial permeability transition (MPT) pore, the goal of this study was to evaluate the therapeutic efficacy of cyclosporin A (CsA), an MPT blocker, to prevent cells from undergoing apoptosis and consequently attenuate late LV remodeling post-MI. MI was induced in C57BL/6 mice and then randomized to either vehicle or CsA groups. Beginning 48 h after surgery after infarction had already occurred, mice were gavaged with CsA (2 mg/kg) or vehicle once daily. LV end-diastolic volume and LV ejection fraction were assessed by echocardiography before MI induction and terminally at either 7 days (n = 7) or 28 days (n = 8) post-MI. LV end-diastolic volume increased and LV ejection fraction decreased in all MI groups with no difference between the CsA-treated and untreated groups. After vehicle and CsA, areas of necrosis were present at 7 and 28 days post-MI with no difference between treatment groups. Caspase-3 activity and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling in distal nonnecrotic LV both increased after MI but were lower in CsA-treated mice compared with vehicle (P < 0.05). In conclusion, CsA decreased apoptosis occurring late after MI, confirming involvement of a CsA-sensitive MPT in the cell death. However, CsA-mediated reduction in apoptosis in non-MI myocardium was not beneficial against late pump dysfunction occurring during post-MI remodeling.

Reproducible Porcine Model of Thoracic Aortic Aneurysm

Background— Thoracic aortic aneurysms (TAAs) develop secondary to abnormal aortic extracellular matrix remodeling, resulting in a weakened and dilated aortic wall that progressed to rupture if left unattended. Currently, no diagnostic/prognostic tests are available for the detection of TAA disease. This is largely driven by the lack of a large animal model, which would permit longitudinal/mechanistic studies. Accordingly, the objective of the present study was to establish a reproducible porcine model of aortic dilatation, which recapitulates the structural and biochemical changes observed during human TAA development. Methods and Results— Descending TAAs were induced in Yorkshire pigs (20–25 kg; n=7) through intra-adventitial injections of collagenase (5 mL, 0.35 mg/mL) and periadventitial application of crystalline CaCl2 (0.5 g). Three weeks after TAA induction, aortas were harvested and tissue was collected for biochemical and histological measurements. A subset of animals underwent MRI preoperatively and at terminal surgery. Results were compared with sham-operated controls (n=6). Three weeks after TAA induction, aortic luminal area increased by 38±13% (P=0.018 versus control). Aortic structural changes included elastic lamellar degradation and decreased collagen content. The protein abundance of matrix metalloproteinases 3, 8, 9, and 12 increased in TAA tissue homogenates, whereas tissue inhibitors of metalloproteinases 1 and 4 decreased. Conclusions— These data demonstrate aortic dilatation, aortic medial degeneration, and alterations in matrix metalloproteinase/tissue inhibitors of metalloproteinase abundance, consistent with TAA formation. This study establishes for the first time a large animal model of TAA that recapitulates the hallmarks of human disease and provides a reproducible test bed for examining diagnostic, prognostic, and therapeutic strategies.

Institution of localized high-frequency electrical stimulation targeting early myocardial infarction: Effects on left ventricle function and geometry

Background Although strategies have focused on myocardial salvage/regeneration in the context of an acute coronary syndrome and a myocardial infarction (MI), interventions targeting the formed MI region and altering the course of the post‐MI remodeling process have not been as well studied. This study tested the hypothesis that localized high‐frequency stimulation instituted within a formed MI region using low‐amplitude electrical pulses would favorably change the trajectory of changes in left ventricle geometry and function. Methods At 7 days following MI induction, pigs were randomized for localized high‐frequency stimulation (n = 5, 240 bpm, 0.8 V, and 0.05 ms pulses) or unstimulated (n = 6). Left ventricle geometry and function were measured at baseline (pre‐MI) and at 7, 14, 21, and 28 days post‐MI using echocardiography. MI size at 28 days post‐MI was determined by histochemical staining and planimetry. Results At 7 days post‐MI and before randomization to localized high‐frequency stimulation, left ventricular ejection fraction and end‐diastolic volume was equivalent. However, when compared with 7‐day post‐MI values, left ventricle end‐diastolic volume increased in a time‐dependent manner in the MI unstimulated group, but the relative increase in left ventricle end‐diastolic volume was reduced in the MI localized high‐frequency stimulation group. For example, by 28 days post‐MI, left ventricle end‐diastolic volume increased by 32% in the MI unstimulated group but only by 12% in the MI localized high‐frequency stimulation group (P < .05). Whereas left ventricular ejection fraction appeared unchanged between MI groups, estimates of pulmonary capillary wedge pressure, a marker of adverse left ventricle performance and progression to failure, increased by 62% in the MI unstimulated group and actually decreased by 17% in the MI localized high‐frequency stimulation group when compared with 7‐day post‐MI values (P < .05). MI size was equivalent between the MI groups, indicative of no difference in the extent of absolute myocardial injury. Conclusions The unique findings from this study are 2‐fold. First, targeting the MI region following the resolution of the acute event using a localized stimulation approach is feasible. Second, localized stimulation modified a key parameter of adverse post‐MI remodeling (dilation) and progression to heart failure. These findings demonstrate that the MI region itself is a modifiable tissue and responsive to localized electrical stimulation.