MO453SERUM AND VASCULAR FIBROBLAST GROWTH FACTOR 23 (FGF23) ARE ASSOCIATED WITH VASCULAR CALCIFICATION

Background and Aims Mineral metabolism imbalances and inflammation are related to the development of vascular calcification (VC). Fibroblast growth factor-23 (FGF23) is the main regulator of phosphate homeostasis and various studies have shown the existence of an association between elevated levels of FGF23 and the appearance of cardiovascular disease (CVD). We conducted a case-control study to test the hypothesis that serum and vascular levels of FGF23 are associated with the presence of VC. In addition, we determined the influence of inflammation in these levels. Method One hundred and thirty-three patients diagnosed with clinical atherosclerotic disease undergoing elective vascular surgery, and 20 cadaveric organ donors with no medical history of CVD, were included in this study. Serum levels of intact FGF23 (iFGF23), together with tumor necrosis factor-alpha (TNFα), interleukin (IL)10, were determined by ELISA. Vascular fragments of aorta, carotid and femoral arteries were obtained for assaying the gene expression of FGF23, TNF, IL10 and RUNX2 by qPCR. Immunohistochemical procedures were employed to determine vascular protein levels of FGF23, TNFα and IL10. VC was diagnosed by imaging techniques and confirmed by histological procedures including von Kossa staining. Results Case group presented a higher prevalence of hypertension, hypercholesterolemia and reduced estimated glomerular filtration rate, with no differences regarding other parameters. Serum iFGF23 and TNFα/IL10 ratio were higher in the case group (P<0.01 and P<0.001, respectively). Vascular expression of FGF23 was detected in 58.6% of CVD patients vs 35% of donors, with mean expression levels significantly higher in the first group (P<0.01). Vascular expression of TNF/IL10 was also increased (P<0.001) in CVD patients. FGF23 immunoreactivity was detected in 84% of CVD patients and only in 35% of controls. Immunoreactivity for FGF23 and TNFα/IL10 ratio were significantly higher in CVD patients (P<0.001 and P<0.0001, respectively). Stratified analysis according to serum iFGF23 levels showed a higher prevalence of VC in the upper tertiles. Patients with VC presented increased levels of all the FGF23 variables including serum [1.5 (1.2-1.6) vs. 1.4 (0.9-1.5) pg/mL, P<0.01], vascular mRNA [26.1 (14.3-67.4) vs. 18.8 (8.8-312.9) log AU, P<0.01] and vascular immunoreactivity [4.6 (3.8-4.9) vs. 3.7 (3.1-4.1) log µm2, P<0.05]. Moreover, FGF23 immunoreactivity was detected in 92.3% of fragments with VC and only in 53.6% of those without VC. Serum TNFα/IL10 and RUNX2 mRNA levels were also higher in this group (P<0.01 for both). Correlation analysis showed associations of serum iFGF23 with serum TNFα (r=0.375, P<0.001), neutrophil/lymphocyte (r=0.142, P<0.05), vascular RUNX2 mRNA (r=0.55, P<0.05), and vascular FGF23 immunoreactivity (r=0.281, P<0.05) in the CVD group. Vascular FGF23 expression correlated with RUNX2 mRNA (r=0.315, P<0.05) and FGF23 immunoreactivity (r=0.254, P<0.05). Multiple regression analysis showed that iFGF23 levels were determined by UAE, HDL, FGe, calcium and TNFα levels (adjusted R2= 0.473, P<0.0001) and that vascular FGF23 mRNA expression was determined by TNFα, PCR, glucose and age (adjusted R2= 0.795, P<0.0001). Multivariate logistic regression, with VC as dependent variable, showed that both iFGF23 and vascular mRNA constitute independent risk factors for the existence of VC [OR (95% CI): 1.05 and 1.12, P<0.05 for both]. Conclusion Patients with atherosclerosis and VC present significantly higher serum concentrations of FGF23, as well as higher immunoreactivity and gene expression levels in the vascular wall compared to patients without VC. Moreover, both serum and vascular mRNA levels of FGF23 are associated with the inflammatory status. Whether the increase in systemic and vascular FGF23 can directly promote or favor the calcifying process in the vascular bed is currently an issue under discussion.

Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive pre-clinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced VEGF-induced permeability and edema formation after intra-ocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation dependent manner and that loss of inner blood-retinal barrier (iBRB) integrity induced by diabetes contributes to vision loss.

Vascular Expression of Permeability-Resistant Occludin Mutant Preserves Visual Function in Diabetes

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive pre-clinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced VEGF-induced permeability and edema formation after intra-ocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation dependent manner and that loss of inner blood-retinal barrier (iBRB) integrity induced by diabetes contributes to vision loss.

The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

There is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer (ΔenFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 – implications for microvascular inflammation and hypercoagulopathy in COVID-19

Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.

Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease

Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification (VC) and requires carboxylation by vitamin K to exert calcification inhibition. Chronic kidney disease (CKD) patients undergo early vascular aging often involving extensive VC. The present cross-sectional study investigated the association between circulating dp-ucMGP levels, MGP expression in vascular tissue and MGP polymorphisms. In 141 CKD stage 5 patients, CAC score was significantly increased in the highest tertile of dp-ucMGP (p = 0.002), and a high medial VC score was associated with elevated dp-ucMGP levels. MGP vascular expression was associated with increased circulating dp-ucMGP and CAC scores. MGP SNP analysis revealed that patients homozygous for the C allele of the rs1800801 variant had a higher CAC score (median 15 [range 0–1312]) compared to patients carrying a T allele (median 0 [range 0–966] AU). These results indicate that plasma levels of dp-ucMGP are an independent predictor of increased VC in CKD5 patients and correlate with both higher CAC scores and degree of medial calcification. Additionally, high vascular expression of MGP was associated with higher CAC scores and plasma dp-ucMGP levels. Taken together, our results support that MGP is involved in the pathogenesis of VC.