Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Recipient Vessel Dissection Technique in Diabetic Foot Patients with Perivascular Fibrosis.

Diabetic foot ulcers are becoming more prevalent, and microsurgical free flaps are gaining popularity in salvaging the foot after postdebridement. The perivascular fibrosis around the recipient vessels often makes the dissection of vessels difficult. In this article, we describe a technique of dissection in such situations where fibrosis and dissection of vessels are fraught with danger. In the period from November 2020 to May 2023, 72 free flap reconstructions of the diabetic foot were done at our institute using this technique, with a success rate of 95.8%.

Melatonin ameliorates PM2.5 -induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis.

Fine particulate matter (PM2.5 ) exposure is correlated with the risk of developing cardiac fibrosis. Melatonin is a major secretory product of the pineal gland that has been reported to prevent fibrosis. However, whether melatonin affects the adverse health effects of PM2.5 exposure has not been investigated. Thus, this study was aimed to investigate the protective effect of melatonin against PM2.5 -accelerated cardiac fibrosis. The echocardiography revealed that PM2.5 had impaired both systolic and diastolic cardiac function in ApoE-/- mice. Histopathological analysis demonstrated that PM2.5 induced cardiomyocyte hypertrophy and fibrosis, particularly perivascular fibrosis, while the melatonin administration was effective in alleviating PM2.5 -induced cardiac dysfunction and fibrosis in mice. Results of electron microscopy and confocal scanning laser microscope confirmed that melatonin had restorative effects against impaired mitochondrial ultrastructure and augmented mitochondrial ROS generation in PM2.5 -treated group. Further investigation revealed melatonin administration could significantly reverse the PM2.5 -induced phenotypic modulation of cardiac fibroblasts into myofibroblasts. For the first time, our study found that melatonin effectively alleviates PM2.5 -induced cardiac dysfunction and fibrosis via inhibiting mitochondrial oxidative injury and regulating SIRT3-mediated SOD2 deacetylation. Our findings indicate that melatonin could be a therapy medicine for prevention and treatment of air pollution-associated cardiac diseases.

Inhibition of miR-21 alleviated cardiac perivascular fibrosis via repressing EndMT in T1DM.

In type 1 and type 2 diabetes mellitus, increased cardiac fibrosis, stiffness and associated diastolic dysfunction may be the earliest pathological phenomena in diabetic cardiomyopathy. Endothelial-mesenchymal transition (EndMT) in endothelia cells (ECs) is a critical cellular phenomenon that increases cardiac fibroblasts (CFs) and cardiac fibrosis in diabetic hearts. The purpose of this paper is to explore the molecular mechanism of miR-21 regulating EndMT and cardiac perivascular fibrosis in diabetic cardiomyopathy. In vivo, hyperglycaemia up-regulated the mRNA level of miR-21, aggravated cardiac dysfunction and collagen deposition. The condition was recovered by inhibition of miR-21 following with improving cardiac function and decreasing collagen deposition. miR-21 inhibition decreased cardiac perivascular fibrosis by suppressing EndMT and up-regulating SMAD7 whereas activating p-SMAD2 and p-SMAD3. In vitro, high glucose (HG) up-regulated miR-21 and induced EndMT in ECs, which was decreased by inhibition of miR-21. A highly conserved binding site of NF-κB located in miR-21 5'-UTR was identified. In ECs, SMAD7 is directly regulated by miR-21. In conclusion, the pathway of NF-κB/miR-21/SMAD7 regulated the process of EndMT in T1DM, in diabetic cardiomyopathy, which may be regarded as a potential clinical therapeutic target for cardiac perivascular fibrosis.

Celecoxib, but not indomethacin, ameliorates the hypertensive and perivascular fibrotic actions of cyclosporine in rats: role of endothelin signaling.

The immunosuppressant drug cyclosporine (CSA) is used with nonsteroidal antiinflammatory drugs (NSAIDs) in arthritic conditions. In this study, we investigated whether NSAIDs modify the deleterious hypertensive action of CSA and the role of endothelin (ET) receptors in this interaction. Pharmacologic, protein expression, and histopathologic studies were performed in rats to investigate the roles of endothelin receptors (ETA/ETB) in the hemodynamic interaction between CSA and two NSAIDs, indomethacin and celecoxib. Tail-cuff plethysmography measurements showed that CSA (20 mg kg(-1) day(-1), 10 days) increased systolic blood pressure (SBP) and heart rate (HR). CSA hypertension was associated with renal perivascular fibrosis and divergent changes in immunohistochemical signals of renal arteriolar ETA (increases) and ETB (decreases) receptors. While these effects of CSA were preserved in rats treated concomitantly with indomethacin (5 mg kg(-1) day(-1)), celecoxib (10 mg kg(-1) day(-1)) abolished the pressor, tachycardic, and fibrotic effects of CSA and normalized the altered renal ETA/ETB receptor expressions. Selective blockade of ETA receptors by atrasentan (5 mg kg(-1) day(-1)) abolished the pressor response elicited by CSA or CSA plus indomethacin. Alternatively, BQ788 (ETB receptor blocker, 0.1 mg kg(-1) day(-1)) caused celecoxib-sensitive elevations in SBP and potentiated the pressor response evoked by CSA. Together, the improved renovascular fibrotic and endothelin receptor profile (ETA downregulation and ETB upregulation) mediate, at least partly, the protective effect of celecoxib against the hypertensive effect of CSA. Clinically, the use of celecoxib along with CSA in the management of arthritic conditions might provide hypertension-free regimen.

A Case of Eosinophilic Angiocentric Fibrosis With Palatal Fistulas.

Eosinophilic angiocentric fibrosis (EAF) is a rare, but benign, tumefactive lesion of the head and neck regions. It was initially discovered in 1983 but has recently been connected to the spectrum of immunoglobulin G4-related disease (IgG4-RD). It commonly presents with symptoms of nasal obstruction, structural deformities of the external nose, and involvement of the nasal septum and lateral nasal wall. Our patient presented with a saddle nose deformity, a septal perforation, and palatal fistulas. Laboratory testing for EAF is often negative for the presence of antinuclear cytoplasmic antibodies (ANCA). A definitive diagnosis of EAF can be made through histopathological analysis of the lesion. The appearance of "onion-skin" fibrosis with perivascular infiltration of primary eosinophils is pathognomonic for EAF. While there is a presence of ulceration tissue, EAF does not have any histological signs of necrosis. EAF is a very uncommon etiology of nasal obstructive symptoms; therefore, it is necessary to rule out more conventional pathologies. Even though it appears as a malignant process, it has an excellent prognosis. The common treatment modalities for an active lesion of EAF involve either surgical resection of margins alone or a combination of corticosteroids and resection. Rituximab has also shown benefits in the management of IgG4-RD as a corticosteroid-sparing treatment. Rituximab was chosen for treatment in our patient because surgical resection was not possible due to the absence of an active lesion. In this article, we provide a brief review of EAF and provide a unique case of EAF presenting with oronasal palatal fistulas.

Repeated Social Defeat Enhances CaCl2-Induced Abdominal Aortic Aneurysm Expansion by Inhibiting the Early Fibrotic Response via the MAPK-MKP-1 Pathway.

Depression is an independent risk factor for cardiovascular disease and is significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the effect of repeated social defeat (RSD) on AAA development. Eight-week-old male wild-type mice were exposed to RSD by being housed with larger CD-1 mice in a shared cage. They were subjected to vigorous physical contact. After the confirmation of depressive-like behavior, calcium chloride was applied to the infrarenal aorta of the mice. At one week, AAA development was comparable between the defeated and control mice, without any differences being observed in the accumulated macrophages or in the matrix metalloproteinase activity. At two weeks, the maximum diameter and circumference of the aneurysm were significantly increased in the defeated mice, and a significant decrease in periaortic fibrosis was also observed. Consistently, the phosphorylation of the extracellular signal-regulated kinase and the incorporation of 5-bromo-2'-deoxyuridine in the primarily cultured aortic vascular smooth muscle cells were significantly reduced in the defeated mice, which was accompanied by a substantial increase in mitogen-activated protein kinase phosphatase-1 (MKP-1). The MKP-1 mRNA and protein expression levels during AAA were much higher in the defeated mice than they were in the control mice. Our findings demonstrate that RSD enhances AAA development by suppressing periaortic fibrosis after an acute inflammatory response and imply novel mechanisms that are associated with depression-related AAA development.

Endothelial cell metabolic memory causes cardiovascular dysfunction in diabetes.

AIMS: The aim of this study was to identify the molecular mechanism for hyperglycaemia-induced metabolic memory in endothelial cells (ECs), and to show its critical importance to development of cardiovascular dysfunction in diabetes. METHODS AND RESULTS: Hyperglycaemia induces increased nuclear factor-κB (NF-κB) signalling, up-regulation of miR-27a-3p, down-regulation of nuclear factor erythroid-2 related factor 2 (NRF2) expression, increased transforming growth factor-ß (TGF-ß) signalling, down-regulation of miR-29, and induction of endothelial-to-mesenchymal transition (EndMT), all of which are memorized by ECs and not erased when switched to a low glucose condition, thereby causing perivascular fibrosis and cardiac dysfunction. Similar metabolic memory effects are found for production of nitric oxide (NO), generation of reactive oxygen species (ROS), and the mitochondrial oxygen consumption rate in two different types of ECs. The observed metabolic memory effects in ECs are blocked by NRF2 activator tert-butylhydroquinone and a miR-27a-3p inhibitor. In vivo, the NRF2 activator and miR-27a-3p inhibitor block cardiac perivascular fibrosis and restore cardiovascular function by decreasing NF-κB signalling, down-regulating miR-27a-3p, up-regulating NRF2 expression, reducing TGF-ß signalling, and inhibiting EndMT during insulin treatment of diabetes in streptozotocin-induced diabetic mice, whereas insulin alone does not improve cardiac function. CONCLUSIONS: Our data indicate that disruption of hyperglycaemia-induced EC metabolic memory is required for restoring cardiac function during treatment of diabetes, and identify a novel molecular signalling pathway of NF-κB/miR-27a-3p/NRF2/ROS/TGF-ß/EndMT involved in metabolic memory.

Irisin ameliorates doxorubicin-induced cardiac perivascular fibrosis through inhibiting endothelial-to-mesenchymal transition by regulating ROS accumulation and autophagy disorder in endothelial cells.

The dose-dependent toxicity to cardiomyocytes has been well recognized as a central characteristic of doxorubicin (DOX)-induced cardiotoxicity (DIC), however, the pathogenesis of DIC in the cardiac microenvironment remains elusive. Irisin is a new hormone-like myokine released into the circulation in response to exercise with distinct functions in regulating apoptosis, inflammation, and oxidative stress. Recent advances revealed the role of irisin as a novel therapeutic method and an important mediator of the beneficial effects of exercise in cardioprotection. Here, by using a low-dose long-term mouse DIC model, we found that the perivascular fibrosis was involved in its myocardial toxicity with the underlying mechanism of endothelial-to-mesenchymal transition (EndMT). Irisin treatment could partially reverse DOX-induced perivascular fibrosis and cardiotoxicity compared to endurance exercise. Mechanistically, DOX stimulation led to excessive accumulation of ROS, which activated the NF-κB-Snail pathway and resulted in EndMT. Besides, dysregulation of autophagy was also found in DOX-treated endothelial cells. Restoring autophagy flux could ameliorate EndMT and eliminate ROS. Irisin treatment significantly alleviated ROS accumulation, autophagy disorder, NF-κB-Snail pathway activation as well as the phenotype of EndMT by targeting uncoupling protein 2 (UCP2). Our results also initially found that irisin was mainly secreted by cardiomyocytes in the cardiac microenvironment, which was significantly reduced by DOX intervention, and had a protective effect on endothelial cells in a paracrine manner. In summary, our study indicated that DOX-induced ROS accumulation and autophagy disorders caused an EndMT in CMECs, which played a role in the perivascular fibrosis of DIC. Irisin treatment could partially reverse this phenomenon by regulating UCP2. Cardiomyocytes were the main source of irisin in the cardiac microenvironment. The current study provides a novel perspective elucidating the pathogenesis and the potential treatment of DIC.

Estrogen Contributions to Microvascular Dysfunction Evolving to Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a syndrome involving microvascular dysfunction. No treatment is available yet and as the HFpEF patient group is expanding due to the aging population, more knowledge on dysfunction of the cardiac microvasculature is required. Endothelial dysfunction, impaired angiogenesis, (perivascular) fibrosis and the pruning of capillaries (rarefaction) may all contribute to microvascular dysfunction in the heart and other organs, e.g., the kidneys. The HFpEF patient group consists mainly of post-menopausal women and female sex itself is a risk factor for this syndrome. This may point toward a role of estrogen depletion after menopause in the development of HFpEF. Estrogens favor the ratio of vasodilating over vasoconstricting factors, which results in an overall lower blood pressure in women than in men. Furthermore, estrogens improve angiogenic capacity and attenuate (perivascular) fibrosis formation. Therefore, we hypothesize that the drop of estrogen levels after menopause contributes to myocardial microvascular dysfunction and renders post-menopausal women more vulnerable for heart diseases that involve the microvasculature. This review provides a detailed summary of molecular targets of estrogen, which might guide future research and treatment options.

Thrombospondin-4 mediates cardiovascular remodelling in angiotensin II-induced hypertension.

Thrombospondin 4 (TSP-4) expression is induced in the heart and vasculature under pathological conditions, including myocardial infarction, myocardial pressure overload, and hypertension. TSP-4 is linked to remodelling processes, where it may affect extracellular matrix protein organization. In previous work, we studied the role of TSP-4 in small arteries during hypertension using Ang II-treated Thrombospondin 4 knockout (Thbs4-/-) mice. We reported increased heart weight, as well as the occurrence of aortic aneurysms in the Ang II-treated Thbs4-/- animals. In the present study, we further characterized the hearts and aortas from these animals. Hypertrophy of cardiomyocytes, together with perivascular fibrosis and inflammation was observed in the Ang II-treated Thbs4-/- hearts. In the aortas, an increase in the aortic wall cross-sectional area (CSA) and wall thickness of the Ang II-treated Thbs4-/- mice was found. More detailed investigation of the Ang II-treated Thbs4-/- aortas also revealed the appearance of aortic dissections in the outer medial layer of the arteries, as well as pronounced inflammation. No differences were found in several other extracellular matrix-related parameters, such as number of elastin breaks or stress-strain relationships. However, at the ultrastructural level, collagen fibers showed alterations in diameter in the media and adventitia of the Ang II-treated Thbs4-/- mice, in the area prone to dissection. In conclusion, we identified TSP-4 as an important protein in the development of cardiac hypertrophy and aortic dissections in Ang II-induced hypertension.

Prolyl 4-Hydroxylase Domain Protein 3 Overexpression Improved Obstructive Sleep Apnea-Induced Cardiac Perivascular Fibrosis Partially by Suppressing Endothelial-to-Mesenchymal Transition.

BACKGROUND: Intermittent hypoxia (IH) induced by obstructive sleep apnea is the key factor involved in cardiovascular fibrosis. Under persistent hypoxia condition, endothelial cells respond by endothelial-to-mesenchymal transition (EndMT), which is associated with cardiovascular fibrosis. Prolyl 4-hydroxylase domain protein 3 (PHD3) is a cellular oxygen sensor and its expression increased in hypoxia. However, its role in obstructive sleep apnea-induced EndMT and cardiovascular fibrosis is still uncertain. We investigated the potential mechanism of obstructive sleep apnea-induced cardiac perivascular fibrosis and the role of PHD3 in it. METHODS AND RESULTS: In vivo, C56BL/6 mice were exposed to IH for 12 weeks. PHD3 expression was changed by lentivirus-mediated short-hairpin PHD3 and lentivirus carrying PHD3 cDNA. EndMT related protein levels, histological and functional parameters were detected after 12 weeks. In vitro, human umbilical vein endothelial cells were treated with IH/short-hairpin PHD3/lentivirus carrying PHD3 cDNA to explore the mechanism of PHD3 in altered function of human umbilical vein endothelial cells. We found that chronic intermittent hypoxia increase PHD3 expression and EndMT. In vivo, IH accelerate cardiac dysfunction and aggravate collagen deposition via the process of EndMT. And, when PHD3 were overexpressed, cardiac dysfunction and collagen excessive deposition were improved. In vitro, IH induced EndMT, which endow human umbilical vein endothelial cells spindle morphology and an enhanced ability to migration and collagen secretion. PHD3 overexpression in cultured human umbilical vein endothelial cells ameliorated IH-induced EndMT through inactivating hypoxia-inducible factor 1 alpha and small mothers against decapentaplegic 2 and 3. CONCLUSIONS: Obstructive sleep apnea-induced cardiac perivascular fibrosis is associated with EndMT, and PHD3 overexpression might be beneficial in the prevention of it by inhibiting EndMT. PHD3 overexpression might have therapeutic potential in the treatment of the disease.

Simvastatin, atorvastatin, and pravastatin equally improve the hemodynamic status of diabetic rats.

AIM: To investigate if the effect of statins improving cardiovascular (CV) status of diabetics is drug-specific or class-dependent, and the underlying mechanisms involved. METHODS: We compared the results of daily administration over a four-week period of a low dose (10 mg/kg per day) of atorvastatin (AV), simvastatin (SV), and pravastatin (PV) on cardiac performance in diabetic rats. Echocardiographic variables were tested, as well as systolic blood pressure (SBP), acetylcholine (ACh)-induced relaxation, plasma cholesterol levels, and perivascular fibrosis. Malondialdehyde (MDA) and 4-hydroxyalkenal (4-HAE), and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) protein levels were also measured in cardiac and aortic homogenates. RESULTS: In untreated diabetic rats, cholesterol levels were higher than in control rats (CT; n = 8, P < 0.05), and the low dose of statins used did not modify these levels. In diabetic rats, SBP was higher than in CT, and was significantly reduced by all three statins (n = 10, P < 0.05). Echocardiographic parameters (EF, SV, and COI) were all lower in untreated diabetic rats than in CT (n = 10, P < 0.05). These CV parameters were equally improved by all three statins. The maximal relaxation (EMax) induced by ACh in aortic ring from diabetic rats was also improved. Moreover, this relaxation was abolished by 1 mmol/L NG-nitro-L-arginine methyl ester, suggesting the involvement of a NO-dependent mechanism. CONCLUSION: AV, SV, and PV are equally effective in improving CV performance in diabetic rats. All tree statins decreased media thickness, perivascular fibrosis, and both MDA and 4-HAE in the aortas of diabetic rats, without affecting eNOS and iNOS protein levels. The observed hemodynamic benefits are cholesterol-independent. These benefits appear to be secondary to the improved endothelial function, and to the reduced vascular tone and remodeling that result from decreased oxidative stress.

Chronic intermittent mental stress promotes atherosclerotic plaque vulnerability, myocardial infarction and sudden death in mice.

Vulnerable atherosclerotic plaques are prone to plaque rupture leading to acute cardiovascular syndromes and death. Elucidating the risk of plaque rupture is important to define better therapeutic or preventive strategies. In the present study, we investigated the effect of chronic intermittent mental stress on atherosclerotic plaque stability and cardiovascular mortality in apolipoprotein E-deficient (ApoE(-/-)) mice with a heterozygous mutation in the fibrillin-1 gene (Fbn1(C1039G+/)(-)). This mouse model displays exacerbated atherosclerosis with spontaneous plaque ruptures, myocardial infarction and sudden death, when fed a Western-type diet (WD). Female ApoE(-/-)Fbn1(C1039G+/-) mice were fed a WD for up to 25 weeks. After 10 weeks WD, mice were divided in a control (n = 27) and mental stress (n = 29) group. The chronic intermittent mental stress protocol consisted of 3 triggers: water avoidance, damp bedding and restraint stress, in a randomly assigned order lasting 6 h every weekday for 15 weeks. Chronic intermittent mental stress resulted in a significant increase in the amount of macrophages in atherosclerotic plaques of the proximal ascending aorta, whereas type I collagen and fibrous cap thickness were decreased. The coronary arteries of mental stress-treated mice showed larger plaques, more stenosis, and an increased degree of perivascular fibrosis. Moreover, myocardial infarctions occurred more frequently in the mental stress group. As compared to the control group, the survival of stressed ApoE(-/-)Fbn1(C1039G+/-) mice decreased from 67% to 52% at 25 weeks WD, presumably due to myocardial infarctions. In conclusion, chronic intermittent mental stress promotes plaque instability, myocardial infarctions, and mortality of ApoE(-/-)Fbn1(C1039G+/-) mice.

Salt loading exacerbates diastolic dysfunction and cardiac remodeling in young female Ren2 rats.

OBJECTIVE: Recent data would suggest pre-menopausal insulin resistant women are more prone to diastolic dysfunction than men, yet it is unclear why. We and others have reported that transgenic (mRen2)27 (Ren2) rats overexpressing the murine renin transgene are insulin resistant due to oxidative stress in insulin sensitive tissues. As increased salt intake promotes inflammation and oxidative stress, we hypothesized that excess dietary salt would promote diastolic dysfunction in transgenic females under conditions of excess tissue Ang II and circulating aldosterone levels. MATERIALS/METHODS: For this purpose we evaluated cardiac function in young female Ren2 rats or age-matched Sprague-Dawley (SD) littermates exposed to a high (4%) salt or normal rat chow intake for three weeks. RESULTS: Compared to SD littermates, at 10weeks of age, female Ren2 rats fed normal chow showed elevations in left ventricular (LV) systolic pressures, LV and cardiomyocyte hypertrophy, and displayed reductions in LV initial filling rate accompanied by increases in 3-nitrotyrosine content as a marker of oxidant stress. Following 3weeks of a salt diet, female Ren2 rats exhibited no further changes in LV systolic pressure, insulin resistance, or markers of hypertrophy but exaggerated increases in type 1 collagen, 3-nitrotryosine content, and diastolic dysfunction. These findings occurred in parallel with ultrastructural findings of pericapillary fibrosis, increased LV remodeling, and mitochondrial biogenesis. CONCLUSION: These data suggest that a diet high in salt in hypertensive female Ren2 rats promotes greater oxidative stress, maladaptive LV remodeling, fibrosis, and associated diastolic dysfunction without further changes in LV systolic pressure or hypertrophy.