Thrombospondin-1 The Good, The Bad, and The Complicated

The incidence of diabetes mellitus has increased exponentially over the past 2 decades, and individuals with diabetes mellitus are at increased risk for and have poor prognosis with cardiovascular disease.¹ In the myocardium, diabetes mellitus enhances fatty acid metabolism, inhibits glucose oxidation, and modifies intracellular signaling, eventually leading to diabetic cardiomyopathy.² Diabetic cardiomyopathy is characterized by increased inflammation and accumulation of the extracellular matrix, microangiopathy, as well as impaired cardiac diastolic or systolic function.³ The matricellular proteins are a family of nonstructural extracellular matrix proteins that relay biological, chemical, and mechanical signals through binding to cell surface receptors, growth factors, proteases, and structural matrix proteins.⁴ At very low expression in the normal heart, most matricelluar proteins are markedly induced in response to injury, indicating roles in the woundhealing response.⁵

Thrombospondin (TSP)-1, a typical matricellular protein, is a secreted, multimodular, calcium-binding glycoprotein.⁴ Diabetic db/db mice show upregulated cardiac TSP-1 expression, indicating potential TSP-1 roles in the remodeling diabetic heart. In this issue of Circulation Research, the Frangogiannis laboratory investigated the impact of TSP-1 deletion on remodeling of the left ventricle (LV) in the setting of diabetic cardiomyopathy.⁶ The db/db TSP-1-null (dbTSP) mice showed weight gain and metabolic function comparable to db/db mice. However, dbTSP animals showed larger LV volume, attenuated LV hypertrophy, and impaired cardiac reserve. The decreased collagen accumulation in dbTSP mice was associated with increased matrix metalloproteinase (MMP)-2 and MMP-9 activities. TSP-1 deletion did not influence the inflammatory response or the transforming growth factor (TGF)-β signaling pathway in db/db mice. In line with in vivo data, in vitro TSP-1 incorporation into collagen pads did not activate TGF-β but suppressed leptin-triggered MMP-2 activation. TSP-1 deletion prevented capillary rarefaction in db/db mice by inhibiting angiopoietin-2 upregulation. In summary, this study illuminated for the first time that key function of TSP-1 in diabetes mellitus–induced cardiomyopathy is to regulate the fibrotic and angiogenic responses (Figure).

Figure. A mechanistic diagram depicting thrombospondin (TSP)-1 regulation of diabetic cardiomyopathy.

In the diabetic setting, TSP-1 deletion increases matrix metalloproteinase (MMP)-2 and MMP-9 activities to degrade collagen and suppress fibroblast function, thereby inhibiting collagen synthesis. Both these processes reduce collagen deposition but also increase left ventricular dimensions. TSP-1 deletion upregulates angiogenesis through inhibiting angiopoetin-2 expression. In addition, TSP-1 deletion leads to attenuated myocardial hypertrophy and impaired cardiac reserve, but the mechanisms remain to be elucidated.

Inflammation plays a critical role in the initiation and progression of diabetic cardiomyopathy. Activated nuclear factor-κB and increased inflammatory mediators have been reported in clinical patients and experimental animals with diabetic cardiomyopathy.^7,8 Unresolved chronic inflammation directly or indirectly triggers myocyte death (both necrosis and apoptosis mechanisms), as well as interstitial and perivascular extracellular matrix deposition, leading to structural and functional impairment.⁹ Anti-inflammatory therapies targeting nuclear factor-κB and proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, have been shown to attenuate cardiac dysfunction in diabetic rodent models.^9,10 In this study, the gene expression of proinflammatory cytokines (eg, tumor necrosis factor-α and interleukin-1β) and chemokines (eg, monocyte chemoattractant protein-1 and interferon γ-induced protein 10), however, did not change in diabetic hearts in the presence or absence of TSP-1. This suggests that the role of inflammation in diabetic cardiomyopathy remains incompletely understood.

Interstitial and perivascular fibrosis are pathological hallmarks of diabetic cardiomyopathy, reflective of increased collagen deposition and crosslinking.² Excessive collagen accumulation promotes myocardial stiffness and diastolic dysfunction. The TGF-β/Smad signaling pathway is activated in experimental models of diabetes mellitus and contributes to cardiac fibrosis by stimulating cardiac fibroblast proliferation and secretion of extracellular matrix, particularly collagen I.¹¹ TSP-1 can activate TGF-β1 by preventing its latency-associated peptide from silencing the mature domain of TGF-β.¹² As such, this links TSP-1 to diabetes mellitus-associated myocardial fibrosis. Consistent with TGF-β1 activation, Smad2/3 signaling was activated and collagen accumulated in the diabetic db/db myocardium. TSP-1 deletion, however, did not affect TGF-β1 signaling, implying that matrix-preserving actions of TSP-1 in the remodeling diabetic heart are not because of TGF-β1 activation. In contrast, dbTSP mice had lower collagen content and higher MMP-2 and MMP-9 activities compared with db/db counterparts, which explained the larger LV volume in the absence of TSP-1. In vitro studies using isolated cardiac fibroblasts validated these findings. However, the underlying mechanisms whereby TSP-1 regulates MMP-2 and MMP-9 activity remain to be elucidated.

Microangiopathy develops in the myocardium of diabetic patients and animals and presents as a thickening of capillary basement membrane, medial thickening of the arteriole, perivascular fibrosis, and decreased capillary density.^13,14 TSP-1 is an endogenous angiostatic factor that inhibits angiogenesis by reducing endothelial cell migration and proliferation, decreasing survival by stimulating apoptosis, and inhibiting nitric oxide and vascular endothelial growth factor signaling.^15,16 In this study, the authors revealed that capillary rarefaction occurred in diabetic db/db mice, an effect abrogated in the absence of TSP-1. The authors demonstrated that the TSP-1 induced angiostatic mechanism involved upregulation of angiopoietin-2 but not vascular endothelial growth factor. Angiopoietin-2 facilitates vessel regression by interrupting Tie2 signaling and pericyte recruitment.¹⁷

The findings of this study are novel and reveal critical roles of TSP-1 in diabetic cardiomyopathy. The following concepts may further elucidate molecular mechanisms of diabetic cardiomyopathy and help in the search for potential therapeutic targets. First, the dbTSP mouse is a systemic deletion model. The impact of TSP-1 deletion on other glucose-metabolizing organs (eg, liver and pancreas) needs to be considered when interpreting the data. The authors showed higher plasma cholesterol levels in dbTSP mice than in db/db counterparts. TSP-1 is overexpressed in hypercholesterolemic arteries, suggesting a close interaction between cholesterol and TSP-1.¹⁸ Whether the increased cholesterol in dbTSP mice contributes to TSP-1 roles in diabetic cardiomyopathy remains to be determined. A discovery proteomics approach would also be useful for providing novel targets affected by TSP-1.¹⁹

Second, the molecular mechanisms of TSP-1 regulation of diabetic cardiomyopathy are not totally understood. How TSP-1 deficiency enhances MMP-2 and MMP-9 activities and downregulates angiopoietin-2 expression need to be investigated further. Additional experiments examining TSP-1 effects to inhibit MMP functions may bring new insights and potential therapeutic approaches.²⁰ The mechanisms whereby TSP-1 deletion attenuates LV hypertrophy and compromises cardiac reserve need to be clarified. Measuring the expression of fetal genes, such as atrial or B-type natriuretic peptides, β-myosin heavy chain, and α-skeletal actin, may help further identify mechanisms.

Third, TSP-1 roles involve multiple components. In the setting of diabetes mellitus, TSP-1 deletion attenuates LV fibrosis, LV hypertrophy, and microvessel rarefaction, and thus is beneficial. At the same time, TSP-1 deletion impairs cardiac reserve and increases LV dimensions, and thus is detrimental. TSP-1-null mice fed a high-fat diet show attenuated adipose inflammation and improved insulin sensitivity.²¹ Endogenous TSP-1 protects the myocardium from myocardial infarction-induced and pressure overload-induced cardiac remodeling.^22,23 Because diabetes mellitus predisposes humans to cardiovascular morbidity and mortality, having a better understanding of how TSP-1 in the diabetic setting responds to stressed conditions (eg, myocardial infarction and hypertension) is warranted.

In conclusion, the Frangogiannis team has revealed direct and indirect effects of TSP-1 on cardiac changes that occur in the setting of diabetes mellitus. This report serves as a foundation for future studies to define the underlying mechanisms of TSP-1 in regulating diabetic cardiomyopathy.