Hyaluronan synthase 3 is protective after cardiac ischemia-reperfusion by preserving the T cell response.

Dysregulated extracellular matrix (ECM) is a hallmark of adverse cardiac remodeling after myocardial infarction (MI). Previous work from our laboratory suggests that synthesis of the major ECM component hyaluronan (HA) may be beneficial for post-infarct healing. Here, we aimed to investigate the mechanisms of hyaluronan synthase 3 (HAS3) in cardiac healing after MI. Mice with genetic deletion of Has3 (Has3 KO) and wildtype mice (WT) underwent 45 min of ischemia with subsequent reperfusion (I/R), followed by monitoring of heart function and analysis of tissue remodeling for up to three weeks. Has3 KO mice exhibited impaired cardiac function as evidenced by a reduced ejection fraction. Accordingly, Has3 deficiency also resulted in an increased scar size. Cardiac fibroblast activation and CD68+ macrophage counts were similar between genotypes. However, we found a significant decrease in CD4 T cells in the hearts of Has3 KO mice seven days post-MI, in particular reduced numbers of CD4+CXCR3+ Th1 and CD4+CD25+Treg cells. Furthermore, Has3 deficient cardiac T cells were less activated and more apoptotic as shown by decreased CD69+ and increased annexin V+ cells, respectively. In vitro assays using activated splenic CD3 T cells demonstrated that Has3 deficiency resulted in reduced expression of the main HA receptor CD44 and diminished T cell proliferation. T cell transendothelial migration was similar between genotypes. Of note, analysis of peripheral blood from patients with ST-elevation myocardial infarction (STEMI) revealed that HAS3 is the predominant HAS isoenzyme also in human T cells. In conclusion, our data suggest that HAS3 is required for mounting a physiological T cell response after MI to support cardiac healing. Therefore, our study may serve as a foundation for the development of novel strategies targeting HA-matrix to preserve T cell function after MI.

SMC-Derived Hyaluronan Modulates Vascular SMC Phenotype in Murine Atherosclerosis.

[Figure: see text].

Basic Biology of Extracellular Matrix in the Cardiovascular System, Part 1/4: JACC Focus Seminar.

The extracellular matrix (ECM) is the noncellular component of tissues in the cardiovascular system and other organs throughout the body. It is formed of filamentous proteins, proteoglycans, and glycosaminoglycans, which extensively interact and whose structure and dynamics are modified by cross-linking, bridging proteins, and cleavage by matrix degrading enzymes. The ECM serves important structural and regulatory roles in establishing tissue architecture and cellular function. The ECM of the developing heart has unique properties created by its emerging contractile nature; similarly, ECM lining blood vessels is highly elastic in order to sustain the basal and pulsatile forces imposed on their walls throughout life. In this part 1 of a 4-part JACC Focus Seminar, we focus on the role, function, and basic biology of the ECM in both heart development and in the adult.

Inhibition of the hyaluronan matrix enhances metabolic anticancer therapy by dichloroacetate in vitro and in vivo.

BACKGROUND AND PURPOSE: Aerobic glycolysis is a unique feature of tumour cells that entails several advantages for cancer progression such as resistance to apoptosis. The low MW compound, dichloroacetate, is a pyruvate dehydrogenase kinase inhibitor, which restores oxidative phosphorylation and induces apoptosis in a variety of cancer entities. However, its therapeutic effectiveness is limited by resistance mechanisms. This study aimed to examine the role of the anti-apoptotic hyaluronan (HA) matrix in this context and to identify a potential add-on treatment option to overcome this limitation. EXPERIMENTAL APPROACH: The metabolic connection between dichloroacetate treatment and HA matrix augmentation was analysed in vitro by quantitative PCR and affinity cytochemistry. Metabolic pathways were analysed using Seahorse, HPLC, fluorophore-assisted carbohydrate electrophoresis, colourimetry, immunoblots, and immunochemistry. The effects of combining dichloroacetate with the HA synthesis inhibitor 4-methylumbelliferone was evaluated in 2D and 3D cell cultures and in a nude mouse tumour xenograft regression model by immunoblot, immunochemistry, and FACS analysis. KEY RESULTS: Mitochondrial reactivation induced by dichloroacetate metabolically activated HA synthesis by augmenting precursors as well as O-GlcNAcylation. This process was blocked by 4-methylumbelliferone, resulting in enhanced anti-tumour efficacy in 2D and 3D cell culture and in a nude mouse tumour xenograft regression model. CONCLUSIONS AND IMPLICATIONS: The HA rich tumour micro-environment represents a metabolic factor contributing to chemotherapy resistance. HA synthesis inhibition exhibited pronounced synergistic actions with dichloroacetate treatment on oesophageal tumour cell proliferation and survival in vitro and in vivo suggesting the combination of these two strategies is an effective anticancer therapy.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Cardiac fibroblast activation and hyaluronan synthesis in response to hyperglycemia and diet-induced insulin resistance.

Diabetic patients are at a greater risk of heart failure due to diabetic cardiomyopathy and worsened outcome post-myocardial infarction. While the molecular mechanisms remain unclear, fibrosis and chronic inflammation are common characteristics of both conditions. Diabetes mellitus (types I and II) results in excessive hyaluronan (HA) deposition in vivo, and hyperglycemia stimulates HA synthesis for several cell types in vitro. HA-rich extracellular matrix contributes to fibrotic, hyperplastic and inflammatory disease progression. We hypothesized that excessive hyperglycemia-driven HA accumulation may contribute to pathological fibroblast activation and fibrotic remodelling in diabetic patients. Therefore, we analysed the impact of both hyperglycemia and diet-induced obesity and insulin resistance on HA matrix formation and cardiac fibroblast activation. Here we report that cardiac fibroblasts isolated from mice on a diabetogenic diet acquire pro-fibrotic gene expression without a concomitant increase in HA matrix deposition. Additionally, hyperglycemia alone does not stimulate HA synthesis or cardiac fibroblast activation in vitro, suggesting that the direct effect of hyperglycemia on fibroblasts is not the primary driver of fibrotic remodelling in cardiac diabetic maladaptation.

Hyperglycaemia and aberrated insulin signalling stimulate tumour progression via induction of the extracellular matrix component hyaluronan.

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose-derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin-induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour-associated HA-matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4-methylumbelliferone, a pharmacological inhibitor of HA-synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long-term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage-independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients.

Deletion of ADAMTS5 does not affect aggrecan or versican degradation but promotes glucose uptake and proteoglycan synthesis in murine adipose derived stromal cells.

ADAMTS5 (TS5), a member of the aggrecanase clade (TS1, 4, 5, 8, 9, 15) of ADAMTS-proteases, has been considered largely responsible for the proteolysis of the hyalectans, aggrecan (Acan) and versican (Vcan), in vivo. However, we have reported that ts5-knockout (KO) mice show joint protection after injury due to inhibition of synovial scarring and enhanced Acan deposition. Also, KO mice have an impaired wound healing phenotype in skin and tendons which is associated with Acan/Vcan-rich deposits at the wound sites. Moreover, the Acan and Vcan deposited was aggrecanase-cleaved, even in the absence of TS5. In this study, we have used adipose-derived stromal cell (ADSC) and epiphyseal chondrocyte cultures from wild type and KO mice to further study the role of TS5 in Acan and Vcan turnover. We have confirmed with both cell types that the aggrecanase-mediated degradation of these hyalectans is not due to TS5, but an aggrecanase which primarily cleaves them before they are secreted. We also provide data which suggests that TS5 protein functions to suppress glucose uptake in ADSCs and thereby inhibits the synthesis, and promotes the intracellular degradation of Acan and Vcan by an ADAMTS other than TS5. We propose that this apparently non-proteolytic role of TS5 explains its anti-chondrogenic and pro-fibrotic effects in murine models of wound repair. A possible role for TS5 in an endocytotic process, involving competitive interactions between TS5, LRP1 and GLUT4 is discussed.

Controlled treadmill exercise eliminates chondroid deposits and restores tensile properties in a new murine tendinopathy model.

Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption and accumulation of a glycosaminoglycan-rich chondroid matrix. Recent clinical reports have illustrated the potential of mechanical loading (exercise) therapies to successfully treat chronic tendinopathies. We have developed a new murine tendinopathy model which requires a single injection of TGF-ß1 into the Achilles tendon midsubstance followed by normal cage activity for 2 weeks. At this time, tendon maximum stress showed a dramatic (66%) reduction relative to that of normal controls and this persisted at four weeks. Loss of material properties was accompanied by abundant chondroid cells within the tendon (closely resembling the changes observed in human samples obtained intra-operatively) and increased expression of Acan, Col1a1, Col2a1, Col3a1, Fn1 and Mmp3. Mice subjected to two weeks of daily treadmill exercise following TGF-ß1 injection showed a similar reduction in tendon material properties as the caged group. However, in mice subjected to 4 weeks of treadmill exercise, tendon maximum stress values were similar to those of naive controls. Tendons from the mice exercised for 4 weeks showed essentially no chondroid cells and the expression of Acan, Col1a1, Col2a1, Col3a1, and Mmp3 was significantly reduced relative to the 4-week cage group. This technically simple murine tendinopathy model is highly amenable to detailed mechanistic and translational studies of the biomechanical and cell biological pathways, that could be targeted to enhance healing of tendinopathy.

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism.

INTRODUCTION: The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. METHODS: The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. RESULTS: Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. CONCLUSIONS: The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.