Pawel Zymek

Critical Role of Endogenous Thrombospondin-1 in Preventing Expansion of Healing Myocardial Infarcts

Background—Matricellular proteins are extracellular matrix proteins that do not contribute directly to tissue integrity but are capable of modulating cell function. We hypothesized that the matricellular protein thrombospondin (TSP)-1, a potent inhibitor of angiogenesis and activator of transforming growth factor (TGF-&bgr;), is induced in healing myocardial infarcts and plays a role in suppressing the postinfarction inflammatory response, inhibiting local angiogenesis, and limiting expansion of granulation tissue into the noninfarcted area. Methods and Results—We used a canine and a murine model of reperfused infarction. TSP-1 mRNA was induced in canine infarcts after 1 hour of ischemia and 3 to 7 days of reperfusion. TSP-1 protein showed a strikingly selective localization in the extracellular matrix, microvascular endothelium, and a subset of mononuclear cells of the infarct border zone after 5 to 28 days of reperfusion. Isolated canine venous endothelial cells showed low-level constitutive expression of TSP-1 mRNA, which was markedly induced by TGF-&bgr;, and basic fibroblast growth factor. Murine infarcts also had marked TSP-1 deposition in the border zone. Infarcted TSP-1−/− mice exhibited sustained upregulation of the chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1&agr;, and interferon-&ggr;–inducible protein-10/CXCL10 and the cytokines interleukin-1&bgr;, interleukin-6, and TGF-&bgr;, suggesting an enhanced and prolonged postinfarction inflammatory response. In addition, TSP-1−/− mice had markedly increased macrophage and myofibroblast density in infarcts and in remodeling noninfarcted myocardial areas neighboring the myocardial scar, suggesting expansion of granulation tissue formation into the noninfarcted territory. TSP-1−/− animals had more extensive postinfarction remodeling than wild-type mice, although infarct size was similar in both groups. Conclusions—The infarct border zone may be capable of modulating the healing process through its unique extracellular matrix content. The selective endogenous expression of TSP-1 in the infarct border zone may serve as a “barrier,” limiting expansion of granulation tissue and protecting the noninfarcted myocardium from fibrotic remodeling.

Extracellular matrix remodeling in canine and mouse myocardial infarcts

Extracellular matrix proteins not only provide structural support, but also modulate cellular behavior by activating signaling pathways. Healing of myocardial infarcts is associated with dynamic changes in the composition of the extracellular matrix; these changes may play an important role in regulating cellular phenotype and gene expression. We examined the time course of extracellular matrix deposition in a canine and mouse model of reperfused infarction. In both models, myocardial infarction resulted in fragmentation and destruction of the cardiac extracellular matrix, extravasation of plasma proteins, such as fibrinogen and fibronectin, and formation of a fibrin-based provisional matrix providing the scaffold for the infiltration of granulation tissue cells. Lysis of the plasma-derived provisional matrix was followed by the formation of a cell-derived network of provisional matrix composed of cellular fibronectin, laminin, and hyaluronic acid and containing matricellular proteins, such as osteopontin and osteonectin/SPARC. Finally, collagen was deposited in the infarct, and the wound matured into a collagen-based scar with low cellular content. Although the canine and mouse infarcts exhibited a similar pattern of extracellular matrix deposition, deposition of the provisional matrix was more transient in the mouse infarct and was followed by earlier formation of a mature collagen-based scar after 7–14 days of reperfusion; at the same timepoint, the canine infarct was highly cellular and evolving. In addition, mature mouse infarcts showed limited collagen deposition and significant tissue loss leading to the formation of a thin scar. In contrast, dogs exhibited extensive collagen accumulation in the infarcted area. These species-specific differences in infarct wound healing should be taken into account when interpreting experimental infarction studies and when attempting to extrapolate the findings to the human pathological process.

CD44 Is Critically Involved in Infarct Healing by Regulating the Inflammatory and Fibrotic Response

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of “stop signals” that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44−/− animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44null infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44null cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-β in comparison to wild-type fibroblasts. The healing defects in CD44−/− mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.

Induction of the CXC Chemokine Interferon-γ–Inducible Protein 10 Regulates the Reparative Response Following Myocardial Infarction

Rationale: Interferon-&ggr;–inducible protein (IP)-10/CXCL10, an angiostatic and antifibrotic chemokine with an important role in T-cell trafficking, is markedly induced in myocardial infarcts, and may regulate the reparative response. Objective: To study the role of IP-10 in cardiac repair and remodeling. Methods and Results: We studied cardiac repair in IP-10–null and wild-type (WT) mice undergoing reperfused infarction protocols and examined the effects of IP-10 on cardiac fibroblast function. IP-10–deficient and WT animals had comparable acute infarct size. However, the absence of IP-10 resulted in a hypercellular early reparative response and delayed contraction of the scar. Infarcted IP-10−/− hearts exhibited accentuated early dilation, followed by rapid wall thinning during infarct maturation associated with systolic dysfunction. Although IP-10–null and WT mice had comparable cytokine expression, the absence of IP-10 was associated with marked alterations in the cellular content of the infarct. IP-10−/− infarcts had more intense infiltration with CD45+ leukocytes, Mac-2+ macrophages, and &agr;-smooth muscle actin (&agr;-SMA)+ myofibroblasts than WT infarcts but exhibited reduced recruitment of the subpopulations of leukocytes, T lymphocytes and &agr;-SMA+ cells that expressed CXCR3, the IP-10 receptor. IP-10 did not modulate cardiac fibroblast proliferation and apoptosis but significantly inhibited basic fibroblast growth factor–induced fibroblast migration. In addition, IP-10 enhanced growth factor–mediated wound contraction in fibroblast-populated collagen lattices. Conclusions: Endogenous IP-10 is an essential inhibitory signal that regulates the cellular composition of the healing infarct and promotes wound contraction, attenuating adverse remodeling. IP-10–mediated actions may be due, at least in part, to direct effects on fibroblast migration and function.

Increased Myocardial Susceptibility to Repetitive Ischemia With High-fat diet-induced Obesity

Obesity and diabetes are frequently associated with cardiovascular disease. When a normal heart is subjected to brief/sublethal repetitive ischemia and reperfusion (I/R), adaptive responses are activated to preserve cardiac structure and function. These responses include but are not limited to alterations in cardiac metabolism, reduced calcium responsiveness, and induction of antioxidant enzymes. In a model of ischemic cardiomyopathy inducible by brief repetitive I/R, we hypothesized that dysregulation of these adaptive responses in diet‐induced obese (DIO) mice would contribute to enhanced myocardial injury. DIO C57BL/6J mice were subjected to 15 min of daily repetitive I/R while under short‐acting anesthesia, a protocol that results in the development of fibrotic cardiomyopathy. Cardiac lipids and candidate gene expression were analyzed at 3 days, and histology at 5 days of repetitive I/R. Total free fatty acids (FFAs) in the cardiac extracts of DIO mice were significantly elevated, reflecting primarily the dietary fatty acid (FA) composition. Compared with lean controls, cardiac FA oxidation (FAO) capacity of DIO mice was significantly higher, concurrent with increased expression of FA metabolism gene transcripts. Following 15 min of daily repetitive I/R for 3 or 5 days, DIO mice exhibited increased susceptibility to I/R and, in contrast to lean mice, developed microinfarction, which was associated with an exaggerated inflammatory response. Repetitive I/R in DIO mice was associated with more profound significant downregulation of FA metabolism gene transcripts and elevated FFAs and triglycerides. Maladaptive metabolic changes of FA metabolism contribute to enhanced myocardial injury in diet‐induced obesity.