Inflammation as A Precursor of Atherothrombosis, Diabetes and Early Vascular Aging

Vascular disease was for a long time considered a disease of the old age, but it is becoming increasingly clear that a cumulus of factors can cause early vascular aging (EVA). Inflammation plays a key role in vascular stiffening and also in other pathologies that induce vascular damage. There is a known and confirmed connection between inflammation and atherosclerosis. However, it has taken a long time to prove the beneficial effects of anti-inflammatory drugs on cardiovascular events. Diabetes can be both a product of inflammation and a cofactor implicated in the progression of vascular disease. When diabetes and inflammation are accompanied by obesity, this ominous trifecta leads to an increased incidence of atherothrombotic events. Research into earlier stages of vascular disease, and documentation of vulnerability to premature vascular disease, might be the key to success in preventing clinical events. Modulation of inflammation, combined with strict control of classical cardiovascular risk factors, seems to be the winning recipe. Identification of population subsets with a successful vascular aging (supernormal vascular aging—SUPERNOVA) pattern could also bring forth novel therapeutic interventions.

Apelin expression deficiency in mice contributes to vascular stiffening by extracellular matrix remodeling of the aortic wall

Numerous recent studies have shown that in the continuum of cardiovascular diseases, the measurement of arterial stiffness has powerful predictive value in cardiovascular risk and mortality and that this value is independent of other conventional risk factors, such as age, cholesterol levels, diabetes, smoking, or average blood pressure. Vascular stiffening is often the main cause of arterial hypertension (AHT), which is common in the presence of obesity. However, the mechanisms leading to vascular stiffening, as well as preventive factors, remain unclear. The aim of the present study was to investigate the consequences of apelin deficiency on the vascular stiffening and wall remodeling of aorta in mice. This factor freed by visceral adipose tissue, is known for its homeostasic role in lipid and vascular metabolisms, or again in inflammation. We compared the level of metabolic markers, inflammation of white adipose tissue (WAT), and aortic wall remodeling from functional and structural approaches in apelin-deficient and wild-type (WT) mice. Apelin-deficient mice were generated by knockout of the apelin gene (APL-KO). From 8 mice by groups, aortic stiffness was analyzed by pulse wave velocity measurements and by characterizations of collagen and elastic fibers. Mann–Whitney statistical test determined the significant data (p < 5%) between groups. The APL-KO mice developed inflammation, which was associated with significant remodeling of visceral WAT, such as neutrophil elastase and cathepsin S expressions. In vitro, cathepsin S activity was detected in conditioned medium prepared from adipose tissue of the APL-KO mice, and cathepsin S activity induced high fragmentations of elastic fiber of wild-type aorta, suggesting that the WAT secretome could play a major role in vascular stiffening. In vivo, remodeling of the extracellular matrix (ECM), such as collagen accumulation and elastolysis, was observed in the aortic walls of the APL-KO mice, with the latter associated with high cathepsin S activity. In addition, pulse wave velocity (PWV) and AHT were increased in the APL-KO mice. The latter could explain aortic wall remodeling in the APL-KO mice. The absence of apelin expression, particularly in WAT, modified the adipocyte secretome and facilitated remodeling of the ECM of the aortic wall. Thus, elastolysis of elastic fibers and collagen accumulation contributed to vascular stiffening and AHT. Therefore, apelin expression could be a major element to preserve vascular homeostasis.

Plasma Lipoprotein(a) Levels as Determinants of Arterial Stiffening in Hypertension

Previous studies have shown that plasma lipoprotein(a) (Lp(a)) plays an important role in the development of hypertensive organ damage. The aim of the present study was to investigate the relationship of Lp(a) with markers of arterial stiffening in hypertension. In 138 essential hypertensive patients free of diabetes, renal failure and cardiovascular complications, we measured plasma lipids and assessed vascular stiffness through the use of pulse wave analysis and calculation of the brachial augmentation index (AIx), and measured the pulse wave velocity (PWV). Plasma Lp(a) levels were significantly and directly related to both AIx (r = 0.490; P < 0.001) and PWV (r = 0.212; P = 0.013). Multiple regression analysis showed that AIx was independently correlated with age, C-reactive protein, and plasma Lp(a) (beta 0.326; P < 0.001), while PWV was independently and directly correlated with age, and inversely with HDL, but not with plasma Lp(a). Logistic regression indicated that plasma Lp(a) could predict an AIx value above the median for the distribution (P = 0.026). Thus, in a highly selective group of patients with hypertension, plasma Lp(a) levels were significantly and directly related to markers of vascular stiffening. Because of the relevance of vascular stiffening to cardiovascular risk, the reduction of Lp(a) levels might be beneficial for cardiovascular protection in patients with hypertension.

Vascular Stiffening Mediated by Rho‐Associated Coiled‐Coil Containing Kinase Isoforms

Background The pathogenesis of vascular stiffening and hypertension is marked by non‐compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho‐associated coiled‐coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice ( Rock1 +/− and Rock2 +/− ) were used to determine the role of ROCK1 and ROCK2 in age‐related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild‐type mice compared with that of Rock1 +/− and Rock2 +/− mice. As a model for age‐related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L‐N ω ‐nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12‐week‐old male Rock1 +/− and Rock2 +/− mice. Similar to advancing age, angiotensin II/L‐N ω ‐nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2 +/− , and to a lesser extent, Rock1 +/− mice. The reduction of aortic stiffening in Rock2 +/− mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor‐β1 or angiotensin II was greatly attenuated in Rock2 −/− mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age‐related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age‐related vascular stiffening.

Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model

Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Limited Effect of 60-Days Strict Head Down Tilt Bed Rest on Vascular Aging

BackgroundCardiovascular risk may be increased in astronauts after long term space flights based on biomarkers indicating premature vascular aging. We tested the hypothesis that 60 days of strict 6° head down tilt bed rest (HDTBR), an established space analog, promotes vascular stiffening and that artificial gravity training ameliorates the response.MethodsWe studied 24 healthy participants (8 women, 24–55 years, BMI = 24.3 ± 2.1 kg/m2) before and at the end of 60 days HDTBR. 16 subjects were assigned to daily artificial gravity. We applied echocardiography to measure stroke volume and isovolumetric contraction time (ICT), calculated aortic compliance (stroke volume/aortic pulse pressure), and assessed aortic distensibility by MRI. Furthermore, we measured brachial-femoral pulse wave velocity (bfPWV) and pulse wave arrival times (PAT) in different vascular beds by blood pressure cuffs and photoplethysmography. We corrected PAT for ICT (cPAT).ResultsIn the pooled sample, diastolic blood pressure (+8 ± 7 mmHg, p &lt; 0.001), heart rate (+7 ± 9 bpm, p = 0.002) and ICT (+8 ± 13 ms, p = 0.036) increased during HDTBR. Stroke volume decreased by 14 ± 15 ml (p = 0.001). bfPWV, aortic compliance, aortic distensibility and all cPAT remained unchanged. Aortic area tended to increase (p = 0.05). None of the parameters showed significant interaction between HDTBR and artificial gravity training.Conclusion60 days HDTBR, while producing cardiovascular deconditioning and cephalad fluid shifts akin to weightlessness, did not worsen vascular stiffness. Artificial gravity training did not modulate the response.

Sex and body mass index dependent associations between serum 25-hydroxyvitamin D and pulse pressure in middle-aged and older US adults

High pulse pressure (PP) is a valid indicator of arterial stiffness. Many studies have reported that vitamin D concentration is inversely associated with vascular stiffening. This association may differ depending on sex and body mass index (BMI). This study investigated the associations between vitamin D and PP and evaluated whether these associations differ according to sex and BMI, using data for individuals aged ≥ 50 years from the National Health and Nutrition Examination Survey (NHANES) 2007–2010. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were used as biomarkers of vitamin D levels. High PP was defined as ≥ 60 mmHg. Total 25(OH)D concentrations were dose-dependently associated with lower odds ratios (ORs) for high PP (p-trend = 0.01), after controlling for sociodemographic, behavioral, and dietary factors. When stratified by sex, there was a dose-dependent association between total 25(OH)D concentrations and lower risk of high PP (p-trend < 0.001) in females, but not in males. When stratified by BMI, there was a dose-dependent association between total 25(OH)D concentrations and lower risk of high PP (p-trend < 0.001) in non-overweight subjects, but not in overweight subjects. Improving the vitamin D status could delay elevation of PP and vascular stiffening in female and non-overweight subjects.

Factors Associated with Mutations: Their Matching Rates to Cardiovascular and Neurological Diseases

Monogenic hypertension is rare and caused by genetic mutations, but whether factors associated with mutations are disease-specific remains uncertain. Given two factors associated with high mutation rates, we tested how many previously known genes match with (i) proximity to telomeres or (ii) high adenine and thymine content in cardiovascular diseases (CVDs) related to vascular stiffening. We extracted genomic information using a genome data viewer. In human chromosomes, 64 of 79 genetic loci involving >25 rare mutations and single nucleotide polymorphisms satisfied (i) or (ii), resulting in an 81% matching rate. However, this high matching rate was no longer observed as we checked the two factors in genes associated with essential hypertension (EH), thoracic aortic aneurysm (TAA), and congenital heart disease (CHD), resulting in matching rates of 53%, 70%, and 75%, respectively. A matching of telomere proximity or high adenine and thymine content projects the list of loci involving rare mutations of monogenic hypertension better than those of other CVDs, likely due to adoption of rigorous criteria for true-positive signals. Our data suggest that the factor–disease matching rate is an accurate tool that can explain deleterious mutations of monogenic hypertension at a >80% match—unlike the relatively lower matching rates found in human genes of EH, TAA, CHD, and familial Parkinson’s disease.

Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization

Medial vascular calcification (mVC) is closely related to cardiovascular disease, especially in patients suffering from chronic kidney disease (CKD). Even after successful kidney transplantation, cardiovascular mortality remains increased. There is evidence that immunosuppressive drugs might influence pathophysiological mechanisms in the vessel wall. Previously, we have shown in vitro that mVC is induced in vascular smooth muscle cells (VSMCs) upon treatment with azathioprine (AZA). This effect was confirmed in the current study in an in vivo rat model treated with AZA for 24 weeks. The calcium content increased in the aortic tissue upon AZA treatment. The pathophysiologic mechanisms involve AZA catabolism to 6-thiouracil via xanthine oxidase (XO) with subsequent induction of oxidative stress. Proinflammatory cytokines, such as interleukin (IL)-1ß and IL-6, increase upon AZA treatment, both systemically and in the aortic tissue. Further, VSMCs show an increased expression of core-binding factor α-1, alkaline phosphatase and osteopontin. As the AZA effect could be decreased in NLRP3−/− aortic rings in an ex vivo experiment, the signaling pathway might be, at least in part, dependent on the NLRP3 inflammasome. Although human studies are necessary to confirm the harmful effects of AZA on vascular stiffening, these results provide further evidence of induction of VSMC calcification under AZA treatment and its effects on vessel structure.

Small Chain Fatty Acid Phenylbutyric Acid Alleviated Inflammation-Induced Endoplasmic Reticulum Stress in Endothelial Cells

Endothelial cells (EC) have dynamic properties and high plasticity in response to microenvironmental change. A proinflammatory cytokine such as tumor necrotizing factor-α (TNF-α) can induce EC phenotype shift to osteoinduction properties by releasing a potent osteogenic cytokine, namely bone morphogenetic protein 2 (BMP2). Normally BMP2 acts as an osteoblast stimulating factor in bone and cartilage tissue. BMP2 activation in vascular tissue will invite osteoblast recruitment and mineralization and generated pathological vascular stiffening and calcification. Recently, endoplasmic reticulum stress (ERS) has been emerging as a new target therapy in many vascular diseases such as vascular stiffening and calcification. Some short-chain fatty acid like 4-phenyl butyric acid has been shown had anti-ERS properties. However, the role of 4-phenyl butyric acid in BMP2 inhibition in endothelial cells is still poorly understood. Hence, we investigated the role of 4-phenyl butyric acid in inflammation-induced BMP2 expression in human vein derived endothelial cells. Endothelial cells obtained from a baby born umbilical vein were cultured and pre-treated with TNF-α (5 ng/ml) as inflammation precondition. Multiple doses of 4-phenyl butyrate acid (4- PBA) 1 nM/mL, 2 nM/mL, and 3 nM/m were used as ERS inhibitors. The expression of two ERS biomarkers, glucose-related protein-8 (GRP78) and activating transcription factor-6 (ATF6), were measured. Statistical analysis was done using one-way ANOVA and Kruskal Wallis tests, and P<0.01 considered as significant. 4- PBA decrease luminal BMP2 at dose one nM/L, GRP78 at dose 1 nM/L, and translocated ATF6 expression at dose 1 nM/L in endothelial culture dose-dependently. Short-chain fatty acid 4-phenylbutyrate acid decreases luminal ERS marker GRP78 and translocated ATF6 expression in endothelial culture. ERS has a role in osteoinductive phenotype shifting in inflammation endothelial cells, which was the novelty of this research. Further research needs to elucidate ERS inhibition in in vivo experiment.