Nadine Nagy

Inhibition of Hyaluronan Synthesis Accelerates Murine Atherosclerosis Novel Insights Into the Role of Hyaluronan Synthesis

Background— Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results— Four-week-old apolipoprotein E–deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions— The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.Background— Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results— Four-week-old apolipoprotein E–deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions— The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis. # Clinical Perspective {#article-title-39}

Hyaluronan and Hyaluronan Binding Proteins Accumulate in both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate with Inflammatory Cells in Insulitis

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Aberrant mural cell recruitment to lymphatic vessels and impaired lymphatic drainage in a murine model of pulmonary fibrosis

Pulmonary fibrosis is a progressive disease with unknown etiology that is characterized by extensive remodeling of the lung parenchyma, ultimately resulting in respiratory failure. Lymphatic vessels have been implicated with the development of pulmonary fibrosis, but the role of the lymphatic vasculature in the pathogenesis of pulmonary fibrosis remains enigmatic. Here we show in a murine model of pulmonary fibrosis that lymphatic vessels exhibit ectopic mural coverage and that this occurs early during the disease. The abnormal lymphatic vascular patterning in fibrotic lungs was driven by expression of platelet-derived growth factor B (PDGF-B) in lymphatic endothelial cells and signaling through platelet-derived growth factor receptor (PDGFR)-β in associated mural cells. Because of impaired lymphatic drainage, aberrant mural cell coverage fostered the accumulation of fibrogenic molecules and the attraction of fibroblasts to the perilymphatic space. Pharmacologic inhibition of the PDGF-B/PDGFR-β signaling axis disrupted the association of mural cells and lymphatic vessels, improved lymphatic drainage of the lung, and prevented the attraction of fibroblasts to the perilymphatic space. Our results implicate aberrant mural cell recruitment to lymphatic vessels in the pathogenesis of pulmonary fibrosis and that the drainage capacity of pulmonary lymphatics is a critical mediator of fibroproliferative changes.

Inhibitory role of the small leucine-rich proteoglycan biglycan in bladder cancer.

Background Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer. Methods and Results For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors—sunitinib and sorafenib—strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies. Conclusion In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization

Significance 4-methylumbelliferone (4-MU) is an oral drug that inhibits synthesis of hyaluronan, an extracellular matrix polymer implicated in autoimmunity. In the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), 4-MU drives the polarization of T cells away from a Th1 phenotype, associated with disease progression, and toward a FoxP3+ regulatory T-cell phenotype, associated with disease prevention. Moreover, 4-MU inhibits the reactive response of astrocytes, immunocompetent resident cells of the CNS, and prevents trafficking of activated T cells to the CNS. These data suggest that the extracellular matrix, and hyaluronan in particular, may be an active contributor to autoimmune pathogenesis in EAE and, furthermore, that 4-MU is a promising strategy for treatment of CNS autoimmunity. The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Cyclooxygenase inhibitors repress vascular hyaluronan-synthesis in murine atherosclerosis and neointimal thickening

Hyaluronan (HA) is a key molecule of the extracellular matrix that is thought to be critically involved in both atherosclerosis and restenosis. Recently, it has been demonstrated that the cyclooxygenase (COX) products, prostacyclin and prostaglandin E2, induce HA synthesis in vitro by transcriptional up‐regulation of HA‐synthase 2 (HAS2) and HAS1. The relative roles in atherosclerotic and restenotic artery disease of tissue specifically expressed COX‐1 and COX‐2 are still under debate. Thus, the present study aimed to investigate the effect of COX isoform inhibition on HA‐accumulation and regulation of HAS isoform expression in two models of pathologic artery remodelling in vivo. Firstly, ApoE‐deficient mice were treated with a prototypic isoform non‐selective inhibitor, indomethacin or with a prototypic COX‐2 selective inhibitor, rofecoxib, for 8 weeks. Aortic HAS mRNA expression and HA‐accumulation in atherosclerotic aortic root lesions were analyzed. Secondly, neointimal hyperplasia was induced by carotid artery ligation in ApoE‐deficient mice on a high fat diet and the effects of the COX inhibitors were determined after 4 weeks of treatment. Intimal HA‐accumulation was markedly reduced in both models by indomethacin and rofecoxib. This coincided with a strong inhibition of HAS1 mRNA expression in both models and with decreased HAS2 mRNA in the aorta of ApoE‐deficient mice. HAS3 was not affected. The repression of HA‐accumulation by both COX‐2 selective and non‐selective COX inhibition implicates COX‐2 in the regulation of HA synthesis via stimulation of HAS1 and HAS2 expression in vivo. Modulation of vascular HA‐accumulation might play a role in chronic effects of COX inhibitors on the progression of atherosclerosis.

Hyaluronan A Mediator of Islet Dysfunction and Destruction in Diabetes

Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.

Deficiency in Lymphotoxin β Receptor Protects From Atherosclerosis in apoE-Deficient Mice

RATIONALE Lymphotoxin β receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1β2, increased Ccl5 mRNA expression. CONCLUSIONS These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

Impact of eNOS-Dependent Oxidative Stress on Endothelial Function and Neointima Formation

AIMS Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia. RESULTS A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only. INNOVATION Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease. CONCLUSION eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation.