RAS protein activator-like 2 (RASAL2) initiates peritubular capillary rarefaction in hypoxic renal interstitial fibrosis.

The progression of chronic kidney disease (CKD) often involves renal interstitial fibrosis (RIF) and subsequent loss of peritubular capillaries (PTCs), which enhances disease severity. Despite advancements in our understanding of fibrosis, effective interventions for reversing capillary loss remain elusive. Notably, RIF exhibits reduced capillary density, whereas renal cell carcinoma (RCC) shows robust angiogenesis under hypoxic conditions. Using RNA sequencing and bioinformatics, we identified differentially expressed genes (DEGs) in hypoxic human renal tubular epithelial cells (HK-2) and renal cancer cells (786-0). Analysis of altered Ras and PI3K/Akt pathways coupled with hub gene investigation revealed RAS protein activator-like 2 (RASAL2) as a key candidate. Subsequent in vitro and in vivo studies confirmed RASAL2's early-stage response in RIF, which reduced with fibrosis progression. RASAL2 suppression in HK-2 cells enhanced angiogenesis, as evidenced by increased proliferation, migration, and branching of human umbilical vein endothelial cells (HUVECs) co-cultured with HK-2 cells. In mice, RASAL2 knockdown improved Vascular endothelial growth factor A (VEGFA) and Proliferating cell nuclear antigen (PCNA) levels in unilateral ureteral occlusion (UUO)-induced fibrosis (compared to wild type). Hypoxia-inducible factor 1 alpha (HIF-1α) emerged as a pivotal mediator, substantiated by chromatin immunoprecipitation (ChIP) sequencing, with its induction linked to activation. Hypoxia increased the production of RASAL2-enriched extracellular vesicles (EVs) derived from tubular cells, which were internalized by endothelial cells, contributing to the exacerbation of PTC loss. These findings underscore RASAL2's role in mediating reduced angiogenesis in RIF and reveal a novel EV-mediated communication between hypoxic tubular- and endothelial cells, demonstrating a complex interplay between angiogenesis and fibrosis in CKD pathogenesis.

mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

MicroRNA-34a-Dependent Attenuation of Angiogenesis in Right Ventricular Failure.

BACKGROUND: The right ventricle (RV) is at risk in patients with complex congenital heart disease involving right-sided obstructive lesions. We have shown that capillary rarefaction occurs early in the pressure-loaded RV. Here we test the hypothesis that microRNA (miR)-34a, which is induced in RV hypertrophy and RV failure (RVF), blocks the hypoxia-inducible factor-1α-vascular endothelial growth factor (VEGF) axis, leading to the attenuated angiogenic response and increased susceptibility to RV failure. METHODS AND RESULTS: Mice underwent pulmonary artery banding to induce RV hypertrophy and RVF. Capillary rarefaction occurred immediately. Although hypoxia-inducible factor-1α expression increased (0.12±0.01 versus 0.22±0.03, P=0.05), VEGF expression decreased (0.61±0.03 versus 0.22±0.05, P=0.01). miR-34a expression was most upregulated in fibroblasts (4-fold), but also in cardiomyocytes and endothelial cells (2-fold). Overexpression of miR-34a in endothelial cells increased cell senescence (10±3% versus 22±2%, P<0.05) by suppressing sirtulin 1 expression, and decreased tube formation by 50% via suppression of hypoxia-inducible factor-1α, VEGF A, VEGF B, and VEGF receptor 2. miR-34a was induced by stretch, transforming growth factor-ß1, adrenergic stimulation, and hypoxia in cardiac fibroblasts and cardiomyocytes. In mice with RVF, locked nucleic acid-antimiR-34a improved RV shortening fraction and survival half-time and restored capillarity and VEGF expression. In children with congenital heart disease-related RVF, RV capillarity was decreased and miR-34a increased 5-fold. CONCLUSIONS: In summary, miR-34a from fibroblasts, cardiomyocytes, and endothelial cells mediates capillary rarefaction by suppressing the hypoxia-inducible factor-1α-VEGF axis in RV hypertrophy/RVF, raising the potential for anti-miR-34a therapeutics in patients with at-risk RVs.

Permissive role of vascular endothelium in fibrosis: focus on the kidney.

Fibrosis, the morphologic end-result of a plethora of chronic conditions and the scorch for organ function, has been thoroughly investigated. One aspect of its development and progression, namely the permissive role of vascular endothelium, has been overshadowed by studies into (myo)fibroblasts and TGF-ß; thus, it is the subject of the present review. It has been established that tensile forces of the extracellular matrix acting on cells are a prerequisite for mechanochemical coupling, leading to liberation of TGF-ß and formation of myofibroblasts. Increased tensile forces are prompted by elevated vascular permeability in response to diverse stressors, resulting in the exudation of fibronectin, fibrinogen/fibrin, and other proteins, all stiffening the extracellular matrix. These processes lead to the development of endothelial cells dysfunction, endothelial-to-mesenchymal transition, premature senescence of endothelial cells, perturbation of blood flow, and gradual obliteration of microvasculature, leaving behind "string" vessels. The resulting microvascular rarefaction is not only a constant companion of fibrosis but also an adjunct mechanism of its progression. The deepening knowledge of the above chain of pathogenetic events involving endothelial cells, namely increased permeability-stiffening of the matrix-endothelial dysfunction-microvascular rarefaction-tissue fibrosis, may provide a roadmap for therapeutic interventions deemed to curtail and reverse fibrosis.

Capillary rarefaction: a missing link in renal and cardiovascular disease?

Patients with chronic kidney disease (CKD) have an increased risk for cardiovascular morbidity and mortality. Capillary rarefaction may be both one of the causes as well as a consequence of CKD and cardiovascular disease. We reviewed the published literature on human biopsy studies and conclude that renal capillary rarefaction occurs independently of the cause of renal function decline. Moreover, glomerular hypertrophy may be an early sign of generalized endothelial dysfunction, while peritubular capillary loss occurs in advanced renal disease. Recent studies with non-invasive measurements show that capillary rarefaction is detected systemically (e.g., in the skin) in individuals with albuminuria, as sign of early CKD and/or generalized endothelial dysfunction. Decreased capillary density is found in omental fat, muscle and heart biopsies of patients with advanced CKD as well as in skin, fat, muscle, brain and heart biopsies of individuals with cardiovascular risk factors. No biopsy studies have yet been performed on capillary rarefaction in individuals with early CKD. At present it is unknown whether individuals with CKD and cardiovascular disease merely share the same risk factors for capillary rarefaction, or whether there is a causal relationship between rarefaction in renal and systemic capillaries. Further studies on renal and systemic capillary rarefaction, including their temporal relationship and underlying mechanisms are needed. This review stresses the importance of preserving and maintaining capillary integrity and homeostasis in the prevention and management of renal and cardiovascular disease.

Thromboxane-induced cerebral microvascular rarefaction predicts depressive symptom emergence in metabolic disease.

Previous studies have suggested that the loss of microvessel density in the peripheral circulation with evolving metabolic disease severity represents a significant contributor to impaired skeletal muscle oxygenation and fatigue-resistance. Based on this and our recent work, we hypothesized that cerebral microvascular rarefaction was initiated from the increased prooxidant and proinflammatory environment with metabolic disease and is predictive of the severity of the emergence of depressive symptoms in obese Zucker rats (OZRs). In male OZR, cerebrovascular rarefaction followed the emergence of elevated oxidant and inflammatory environments characterized by increased vascular production of thromboxane A2 (TxA2). The subsequent emergence of depressive symptoms in OZR was associated with the timing and severity of the rarefaction. Chronic intervention with antioxidant (TEMPOL) or anti-inflammation (pentoxifylline) therapy blunted the severity of rarefaction and depressive symptoms, although the effectiveness was limited. Blockade of TxA2 production (dazmegrel) or action (SQ-29548) resulted in a stronger therapeutic effect, suggesting that vascular production and action represent a significant contributor to rarefaction and the emergence of depressive symptoms with chronic metabolic disease (although other pathways clearly contribute as well). A de novo biosimulation of cerebrovascular oxygenation in the face of progressive rarefaction demonstrates the increased probability of generating hypoxic regions within the microvascular networks, which could contribute to impaired neuronal metabolism and the emergence of depressive symptoms. The results of the present study also implicate the potential importance of aggressive prodromic intervention in reducing the severity of chronic complications arising from metabolic disease.NEW & NOTEWORTHY With clinical studies linking vascular disease risk to depressive symptom emergence, we used obese Zucker rats, a model of chronic metabolic disease, to identify potential mechanistic links between these two negative outcomes. Depressive symptom severity correlated with the extent of cerebrovascular rarefaction, after increased vascular oxidant stress/inflammation and TxA2 production. Anti-TxA2 interventions prevasculopathy blunted rarefaction and depressive symptoms, while biosimulation indicated that cerebrovascular rarefaction increased hypoxia within capillary networks as a potential contributing mechanism.

Spatiotemporal signaling underlies progressive vascular rarefaction in myocardial infarction.

Therapeutic angiogenesis represents a promising avenue to revascularize the ischemic heart. Its limited success is partly due to our poor understanding of the cardiac stroma, specifically mural cells, and their response to ischemic injury. Here, we combine single-cell and positional transcriptomics to assess the behavior of mural cells within the healing heart. In response to myocardial infarction, mural cells adopt an altered state closely associated with the infarct and retain a distinct lineage from fibroblasts. This response is concurrent with vascular rarefaction and reduced vascular coverage by mural cells. Positional transcriptomics reveals that the infarcted heart is governed by regional-dependent and temporally regulated programs. While the remote zone acts as an important source of pro-angiogenic signals, the infarct zone is accentuated by chronic activation of anti-angiogenic, pro-fibrotic, and inflammatory cues. Together, our work unveils the spatiotemporal programs underlying cardiac repair and establishes an association between vascular deterioration and mural cell dysfunction.

Identifying key genes related to the peritubular capillary rarefaction in renal interstitial fibrosis by bioinformatics.

Renal interstitial fibrosis (RIF) is a key feature of progressive chronic kidney disease (CKD), characterized by tubular epithelial cell (TEC) hypoxia and peritubular capillary (PTC) rarefaction. However, the mechanisms underlying these processes remain poorly understood. To address this knowledge gap, we conducted a comparative transcriptome analysis of hypoxic and normoxic HK-2 cells, identifying 572 differentially expressed genes (DEGs). Subsequent Gene Ontology (GO), protein‒protein interaction (PPI) network, and hub gene analyses revealed significant enrichment of DEGs in the HIF-1 signaling pathway based on KEGG enrichment analysis. To further explore TEC modulation under hypoxic conditions, we performed chromatin immunoprecipitation (ChIP) sequencing targeting HIF-1α, identifying 2915 genes potentially regulated by HIF-1α. By comparing RNA sequencing and ChIP sequencing data, we identified 43 overlapping DEGs. By performing GO analysis and peak annotation with IGV, we identified two candidate molecules, VEGFA and BTG1, that are associated with angiogenesis and whose gene sequences were reliably bound by HIF-1α. Our study elucidates the molecular mechanisms underlying RIF, providing valuable insights for potential therapeutic interventions.

Innate Sleep Apnea in Spontaneously Hypertensive Rats Is Associated With Microvascular Rarefaction and Neuronal Loss in the preBötzinger Complex.

BACKGROUND: Sleep apnea (SA) is a major threat to physical health and carries a significant economic burden. These impacts are worsened by its interaction with, and induction of, its comorbidities. SA holds a bidirectional relationship with hypertension, which drives atherosclerosis/arteriolosclerosis, ultimately culminating in vascular dementia. METHODS: To enable a better understanding of these sequelae of events, we investigated innate SA and its effects on cognition in adult-aged spontaneously hypertensive rats, which have a range of cardiovascular disorders: plethysmography and electroencephalographic/electromyographic recordings were used to assess sleep-wake state, breathing parameters, and sleep-disordered breathing; immunocytochemistry was used to assess vascular and neural health; the forced alteration Y maze and Barnes maze were used to assess short- and long-term memories, respectively; and an anesthetized preparation was used to assess baroreflex sensitivity. RESULTS: Spontaneously hypertensive rats displayed a higher degree of sleep-disordered breathing, which emanates from poor vascular health leading to a loss of preBötzinger Complex neurons. These rats also display small vessel white matter disease, a form of vascular dementia, which may be exacerbated by the SA-induced neuroinflammation in the hippocampus to worsen the related deficits in both long- and short-term memories. CONCLUSIONS: Therefore, we postulate that hypertension induces SA through vascular damage in the respiratory column, culminating in neuronal loss in the inspiratory oscillator. This induction of SA, which, in turn, will independently exacerbate hypertension and neural inflammation, increases the rate of vascular dementia.

Semaphorin3A Exacerbates Cardiac Microvascular Rarefaction in Pressure Overload-Induced Heart Disease.

Microvascular endothelial cells (MiVECs) impair angiogenic potential, leading to microvascular rarefaction, which is a characteristic feature of chronic pressure overload-induced cardiac dysfunction. Semaphorin3A (Sema3A) is a secreted protein upregulated in MiVECs following angiotensin II (Ang II) activation and pressure overload stimuli. However, its role and mechanism in microvascular rarefaction remain elusive. The function and mechanism of action of Sema3A in pressure overload-induced microvascular rarefaction, is explored, through an Ang II-induced animal model of pressure overload. RNA sequencing, immunoblotting analysis, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunofluorescence staining results indicate that Sema3A is predominantly expressed and significantly upregulated in MiVECs under pressure overload. Immunoelectron microscopy and nano-flow cytometry analyses indicate small extracellular vesicles (sEVs), with surface-attached Sema3A, to be a novel tool for efficient release and delivery of Sema3A from the MiVECs to extracellular microenvironment. To investigate pressure overload-mediated cardiac microvascular rarefaction and cardiac fibrosis in vivo, endothelial-specific Sema3A knockdown mice are established. Mechanistically, serum response factor (transcription factor) promotes the production of Sema3A; Sema3A-positive sEVs compete with vascular endothelial growth factor A to bind to neuropilin-1. Therefore, MiVECs lose their ability to respond to angiogenesis. In conclusion, Sema3A is a key pathogenic mediator that impairs the angiogenic potential of MiVECs, which leads to cardiac microvascular rarefaction in pressure overload-induced heart disease.

Mice with endothelial cell-selective adhesion molecule deficiency develop coronary microvascular rarefaction and left ventricle diastolic dysfunction.

Endothelial cell-selective adhesion molecule (ESAM) regulates inflammatory cell adhesion and transmigration and promotes angiogenesis. Here, we examined the role of ESAM in cardiac vascularization, inflammatory cell infiltration, and left ventricle (LV) diastolic function under basal and hemodynamic stress conditions. We employed mice with homozygous genetic deletion of ESAM (ESAM-/- ) and also performed uninephrectomy and aldosterone infusion (UNX-Aldo) to induce volume and pressure overload. Using echocardiography, we found that ESAM-/- mice display no change in systolic function. However, they develop LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E = early, A = late mitral inflow peak velocities), increased E/e' ratio, isovolumic relaxation time (IVRT), and E wave deceleration time. An unbiased automated tracing and 3D reconstruction of coronary vasculature revealed that ESAM-/- mice had reduced coronary vascular density. Arteries of ESAM-/- mice exhibited impaired endothelial sprouting and in cultured endothelial cells siRNA-mediated ESAM knockdown reduced tube formation. Changes in ESAM-/- mice were accompanied by elevated myocardial inflammatory cytokine and myeloperoxidase-positive neutrophil levels. Furthermore, UNX-Aldo procedure in wild type mice induced LV diastolic dysfunction, which was accompanied by significantly increased serum ESAM levels. When compared to wild types, ESAM-/- mice with UNX-Aldo displayed worsening of LV diastolic function, as indicated by increased IVRT and pulmonary edema. Thus, we propose that ESAM plays a mechanistic role in proper myocardial vascularization and the maintenance of LV diastolic function under basal and hemodynamic stress conditions.

Assessment of peritubular capillary rarefaction in kidneys of cats with chronic kidney disease.

BACKGROUND: Hypoxia is a key driver of fibrosis and is associated with capillary rarefaction in humans. OBJECTIVES: Characterize capillary rarefaction in cats with chronic kidney disease (CKD). ANIMALS: Archival kidney tissue from 58 cats with CKD, 20 unaffected cats. METHODS: Cross-sectional study of paraffin-embedded kidney tissue utilizing CD31 immunohistochemistry to highlight vascular structures. Consecutive high-power fields from the cortex (10) and corticomedullary junction (5) were digitally photographed. An observer counted and colored the capillary area. Image analysis was used to determine the capillary number, average capillary size, and average percent capillary area in the cortex and corticomedullary junction. Histologic scoring was performed by a pathologist masked to clinical data. RESULTS: Percent capillary area (cortex) was significantly lower in CKD (median 3.2, range, 0.8-5.6) compared to unaffected cats (4.4, 1.8-7.0; P = <.001) and was negatively correlated with serum creatinine concentrations (r = -.36, P = .0013), glomerulosclerosis (r = -0.39, P = <.001), inflammation (r = -.30, P = .009), and fibrosis (r = -.30, P = .007). Capillary size (cortex) was significantly lower in CKD cats (2591 pixels, 1184-7289) compared to unaffected cats (4523 pixels, 1801-7618; P = <.001) and was negatively correlated with serum creatinine concentrations (r = -.40, P = <.001), glomerulosclerosis (r = -.44, P < .001), inflammation (r = -.42, P = <.001), and fibrosis (r = -.38, P = <.001). CONCLUSIONS AND CLINICAL IMPORTANCE: Capillary rarefaction (decrease in capillary size and percent capillary area) is present in kidneys of cats with CKD and is positively correlated with renal dysfunction and histopathologic lesions.

VEGF (Vascular Endothelial Growth Factor) Inhibition and Hypertension: Does Microvascular Rarefaction Play a Role?

Drugs acting by inhibition of the angiogenic action of VEGF (vascular endothelial growth factor) have become major instruments in the treatment of cancer. The downside of their favorable effects in cancer treatment is their frequent cardiovascular side effects. The most consistent finding thus far on the cardiovascular side effects of VEGF inhibitors is the high incidence of hypertension. In this short review, we discuss the evidence that hypertension occurring during VEGF inhibitor treatment is caused by microvascular rarefaction. After a review of the role of VEGF in microvascular growth and differentiation, we present evidence from studies in experimental models of hypertension as well as clinical studies on the microvascular network changes during and after VEGF inhibitor treatment.

The single-chain relaxin mimetic, B7-33, maintains the cardioprotective effects of relaxin and more rapidly reduces left ventricular fibrosis compared to perindopril in an experimental model of cardiomyopathy.

The hormone, relaxin (RLX), exerts various organ-protective effects independently of etiology. However, its complex two-chain and three disulphide bonded structure is a limitation to its preparation and affordability. Hence, a single chain-derivative of RLX, B7-33, was developed and shown to retain the anti-fibrotic effects of RLX in vitro and in vivo. Here, we determined whether B7-33 could retain the other cardioprotective effects of RLX, and also compared its therapeutic efficacy to the ACE inhibitor, perindopril. Adult male 129sv mice were subjected to isoprenaline (ISO; 25 mg/kg/day, s.c)-induced cardiomyopathy, then s.c-treated with either RLX (0.5 mg/kg/day), B7-33 (0.25 mg/kg/day; equivalent dose corrected for MW) or perindopril (1 mg/kg/day) from days 7-14 post-injury. Control mice received saline instead of ISO. Changes in animal body weight (BW) and systolic blood pressure (SBP) were measured weekly, whilst cardiomyocyte hypertrophy and measures of vascular dysfunction and rarefaction, left ventricular (LV) inflammation and fibrosis were assessed at day 14 post-injury. ISO-injured mice had significantly increased LV inflammation, cardiomyocyte hypertrophy, fibrosis, vascular rarefaction and aortic contractility in the absence of any changes in BW or SBP at day 14 post-injury. Both B7-33 and RLX equivalently reduced LV fibrosis and normalised the ISO-induced LV inflammation and cardiomyocyte hypertrophy, whilst restoring blood vessel density and aortic contractility. Comparatively, perindopril lowered SBP and the ISO-induced LV inflammation and vascular rarefaction, but not fibrosis or hypertrophy. As B7-33 retained the cardioprotective effects of RLX and provided rapid-occurring anti-fibrotic effects compared to perindopril, it could be considered as a cost-effective cardioprotective therapy.

Endothelial cells overexpressing CXCR1/2 are renoprotective in rats with acute kidney injury.

Inflammation that develops with the release of chemokines and cytokines during acute kidney injury (AKI) has been shown to participate in functional renal recovery. Although a major research focus has been on the role of macrophages, the family of C-X-C motif chemokines that promote neutrophil adherence and activation also increases with kidney ischemia-reperfusion (I/R) injury. This study tested the hypothesis that intravenous delivery of endothelial cells (ECs) that overexpress (C-X-C motif) chemokine receptors 1 and 2 (CXCR1 and CXCR2, respectively) improves outcomes in kidney I/R injury. Overexpression of CXCR1/2 enhanced homing of endothelial cells to I/R-injured kidneys and limited interstitial fibrosis, capillary rarefaction, and tissue injury biomarkers (serum creatinine concentration and urinary kidney injury molecule-1) following AKI and also reduced expression of P-selectin and the rodent (C-X-C motif) chemokine cytokine-induced neutrophil chemoattractant (CINC)-2ß as well as the number of myeloperoxidase-positive cells in the postischemic kidney. The serum chemokine/cytokine profile, including CINC-1, showed similar reductions. These findings were not observed in rats given endothelial cells transduced with an empty adenoviral vector (null-ECs) or a vehicle alone. These data indicate that extrarenal endothelial cells that overexpress CXCR1 and CXCR2, but not null-ECs or vehicle alone, reduce I/R kidney injury and preserve kidney function in a rat model of AKI.NEW & NOTEWORTHY Inflammation facilitates kidney ischemia-reperfusion (I/R) injury. Endothelial cells (ECs) that were modified to overexpress (C-X-C motif) chemokine receptor (CXCR)1/2 (CXCR1/2-ECs) were injected immediately following kidney I/R injury. The interaction of CXCR1/2-ECs, but not ECs transduced with an empty adenoviral vector, with injured kidney tissue preserved kidney function and reduced production of inflammatory markers, capillary rarefaction, and interstitial fibrosis. The study highlights a functional role for the C-X-C chemokine pathway in kidney damage following I/R injury.

Thymosin ß4 and MRTF-A mitigate vessel regression despite cardiovascular risk factors.

Since clinical revascularization techniques of coronary or peripheral artery disease (CAD/PAD) focus on macrovessels of the heart, the microcirculatory compartment largely goes unnoticed. However, cardiovascular risk factors not only drive large vessel atherosclerosis, but also microcirculatory rarefaction, an instance unmet by current therapeutic schemes. Angiogenic gene therapy has the potential to reverse capillary rarefaction, but only if the disease-causing inflammation and vessel-destabilization are addressed. This review summarizes the current knowledge with regard to capillary rarefaction due to cardiovascular risk factors. Moreover, the potential of Thymosin ß4 (Tß4) and its downstream signal, myocardin-related transcription factor-A (MRTF-A), to counteract capillary rarefaction are discussed.

Evaluation of Microvascular Rarefaction in Vascular Cognitive Impairment and Heart Failure (CRUCIAL): Study Protocol for an Observational Study.

INTRODUCTION: Microvascular rarefaction, the functional reduction in perfused microvessels and structural reduction of microvascular density, seems to be an important mechanism in the pathophysiology of small blood vessel-related disorders including vascular cognitive impairment (VCI) due to cerebral small vessel disease and heart failure with preserved ejection fraction (HFpEF). Both diseases share common risk factors including hypertension, diabetes mellitus, obesity, and ageing; in turn, these comorbidities are associated with microvascular rarefaction. Our consortium aims to investigate novel non-invasive tools to quantify microvascular health and rarefaction in both organs, as well as surrogate biomarkers for cerebral and/or cardiac rarefaction (via sublingual capillary health, vascular density of the retina, and RNA content of circulating extracellular vesicles), and to determine whether microvascular density relates to disease severity. METHODS: The clinical research program of CRUCIAL consists of four observational cohort studies. We aim to recruit 75 VCI patients, 60 HFpEF patients, 60 patients with severe aortic stenosis (AS) undergoing surgical aortic valve replacement as a pressure overload HFpEF model, and 200 elderly participants with mixed comorbidities to serve as controls. Data collected will include medical history, physical examination, cognitive testing, advanced brain and cardiac MRI, ECG, echocardiography, sublingual capillary health, optical coherence tomography angiography (OCTa), extracellular vesicles RNA analysis, and myocardial remodelling-related serum biomarkers. The AS cohort undergoing surgery will also have myocardial biopsy for histological microvascular assessment. DISCUSSION: CRUCIAL will examine the pathophysiological role of microvascular rarefaction in VCI and HFpEF using advanced brain and cardiac MRI techniques. Furthermore, we will investigate surrogate biomarkers for non-invasive, faster, easier, and cheaper assessment of microvascular density since these are more likely to be disseminated into widespread clinical practice. If microvascular rarefaction is an early marker of developing small vessel diseases, then measuring rarefaction may allow preclinical diagnosis, with implications for screening, risk stratification, and prevention. Further knowledge of the relevance of microvascular rarefaction and its underlying mechanisms may provide new avenues for research and therapeutic targets.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.