Hemodynamic regulation allows stable growth of microvascular networks.

How do vessels find optimal radii? Capillaries are known to adapt their radii to maintain the shear stress of blood flow at the vessel wall at a set point, yet models of adaptation purely based on average shear stress have not been able to produce complex loopy networks that resemble real microvascular systems. For narrow vessels where red blood cells travel in a single file, the shear stress on vessel endothelium peaks sharply when a red blood cell passes through. We show that stable shear-stress-based adaptation is possible if vessel shear stress set points are cued to the stress peaks. Model networks that respond to peak stresses alone can quantitatively reproduce the observed zebrafish trunk microcirculation, including its adaptive trajectory when hematocrit changes or parts of the network are amputated. Our work reveals the potential for mechanotransduction alone to generate stable hydraulically tuned microvascular networks.

Necessary Role of Ceramides in the Human Microvascular Endothelium During Health and Disease.

BACKGROUND: Elevated plasma ceramides and microvascular dysfunction both independently predict adverse cardiac events. Despite the known detrimental effects of ceramide on the microvasculature, evidence suggests that activation of the shear-sensitive, ceramide-forming enzyme NSmase (neutral sphingomyelinase) elicits formation of vasoprotective nitric oxide (NO). Here, we explore a novel hypothesis that acute ceramide formation through NSmase is necessary for maintaining NO signaling within the human microvascular endothelium. We further define the mechanism through which ceramide exerts beneficial effects and discern key mechanistic differences between arterioles from otherwise healthy adults (non-coronary artery disease [CAD]) and patients diagnosed with CAD. METHODS: Human arterioles were dissected from discarded surgical adipose tissue (n=166), and vascular reactivity to flow and C2-ceramide was assessed. Shear-induced NO and mitochondrial hydrogen peroxide (H2O2) production were measured in arterioles using fluorescence microscopy. H2O2 fluorescence was assessed in isolated human umbilical vein endothelial cells. RESULTS: Inhibition of NSmase in arterioles from otherwise healthy adults induced a switch from NO to NOX-2 (NADPH-oxidase 2)-dependent H2O2-mediated flow-induced dilation. Endothelial dysfunction was prevented by treatment with sphingosine-1-phosphate (S1P) and partially prevented by C2-ceramide and an agonist of S1P-receptor 1 (S1PR1); the inhibition of the S1P/S1PR1 signaling axis induced endothelial dysfunction via NOX-2. Ceramide increased NO production in arterioles from non-CAD adults, an effect that was diminished with inhibition of S1P/S1PR1/S1P-receptor 3 signaling. In arterioles from patients with CAD, inhibition of NSmase impaired the overall ability to induce mitochondrial H2O2 production and subsequently dilate to flow, an effect not restored with exogenous S1P. Acute ceramide administration to arterioles from patients with CAD promoted H2O2 as opposed to NO production, an effect dependent on S1P-receptor 3 signaling. CONCLUSION: These data suggest that despite differential downstream signaling between health and disease, NSmase-mediated ceramide formation is necessary for proper functioning of the human microvascular endothelium. Therapeutic strategies that aim to significantly lower ceramide formation may prove detrimental to the microvasculature.

3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

BACKGROUND: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. METHODS: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. RESULTS: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. CONCLUSIONS: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

Ying and Yang of Ceramide in the Vascular Endothelium.

Ceramides, a group of biologically active sphingolipids, have been described as the new cholesterol given strong evidence linking high plasma ceramide with endothelial damage, risk for early adverse cardiovascular events, and development of cardiometabolic disease. This relationship has sparked great interest in investigating therapeutic targets with the goal of suppressing ceramide formation. However, the growing data challenge this paradigm of ceramide as solely eliciting detrimental effects to the cardiovascular system. Studies show that ceramides are necessary for maintaining proper endothelial redox states, mechanosensation, and membrane integrity. Recent work in preclinical models and isolated human microvessels highlights that the loss of ceramide formation can in fact propagate vascular endothelial dysfunction. Here, we delve into these conflicting findings to evaluate how ceramide may be capable of exerting both beneficial and damaging effects within the vascular endothelium. We propose a unifying theory that while basal levels of ceramide in response to physiological stimuli are required for the production of vasoprotective metabolites such as S1P (sphingosine-1-phosphate), the chronic accumulation of ceramide can promote activation of pro-oxidative stress pathways in endothelial cells. Clinically, the evidence discussed here highlights the potential challenges associated with therapeutic suppression of ceramide formation as a means of reducing cardiovascular disease risk.

Microvascular Network Remodeling in the Ischemic Mouse Brain Defined by Light Sheet Microscopy.

BACKGROUND: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges. METHODS: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling. RESULTS: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug. CONCLUSIONS: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field.

Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

Hemodynamic Characteristics of a Tortuous Microvessel Using High-Fidelity Red Blood Cell Resolved Simulations.

OBJECTIVE: Tortuous microvessels are characteristic of microvascular remodeling associated with numerous physiological and pathological scenarios. Three-dimensional (3D) hemodynamics in tortuous microvessels influenced by red blood cells (RBCs), however, are largely unknown, and important questions remain. Is blood viscosity influenced by vessel tortuosity? How do RBC dynamics affect wall shear stress (WSS) patterns and the near-wall cell-free layer (CFL) over a range of conditions? The objective of this work was to parameterize hemodynamic characteristics unique to a tortuous microvessel. METHODS: RBC-resolved simulations were performed using an immersed boundary method-based 3D fluid dynamics solver. A representative tortuous microvessel was selected from a stimulated angiogenic network obtained from imaging of the rat mesentery and digitally reconstructed for the simulations. The representative microvessel was a venule with a diameter of approximately 20 µm. The model assumes a constant diameter along the vessel length and does not consider variations due to endothelial cell shapes or the endothelial surface layer. RESULTS: Microvessel tortuosity was observed to increase blood apparent viscosity compared to a straight tube by up to 26%. WSS spatial variations in high curvature regions reached 23.6 dyne/cm2 over the vessel cross-section. The magnitudes of WSS and CFL thickness variations due to tortuosity were strongly influenced by shear rate and negligibly influenced by tube hematocrit levels. CONCLUSIONS: New findings from this work reveal unique tortuosity-dependent hemodynamic characteristics over a range of conditions. The results provide new thought-provoking information to better understand the contribution of tortuous vessels in physiological and pathological processes and help improve reduced-order models.

Handheld vital microscopy for the identification of microcirculatory alterations in cervical intraepithelial neoplasia and cervical cancer.

BACKGROUND: Ninety percent of cervical cancer (CC) diagnoses and deaths occur in low and middle-income countries (LMICs). Especially in these countries, where human and material resources are limited, there is a need for real-time screening methods that enable immediate treatment decisions (i.e., 'see and treat'). OBJECTIVE: To evaluate whether handheld vital microscopy (HVM) enables real-time detection of microvascular alterations associated with cervical intraepithelial neoplasia (CIN) and CC. METHODS: A cross-sectional study was conducted in an oncologic hospital and outpatient clinic, and included ten healthy controls, ten women with CIN, and ten women with CC. The microvasculature was assessed in four quadrants of the uterine cervix using HVM. The primary outcome was the presence of abnormal angioarchitecture (AA). Secondary outcomes included capillary loop density (CD), total vessel density (TVD), functional capillary density (FCD), and the proportion of perfused vessels (PPV). RESULTS: 198 image sequences of the cervical microvasculature were recorded. Compared to healthy controls, significantly more abnormal image sequences were observed in women with high-grade CIN (11 % vs. 44 %, P < 0.001) and women with CC (11 % vs. 69 %, P < 0.001). TVD, FCD, and PPV were lower in women with CIN and CC. CONCLUSIONS: HVM enables easy, real-time, non-invasive assessment of cervical lesions through the detection of microvascular alterations. Thereby, HVM potentially provides an opportunity for point-of-care screening, which may enable immediate treatment decisions (see and treat) and reduce the number of unnecessary surgical interventions.

Microvascular smooth muscle cells exhibit divergent phenotypic switching responses to platelet-derived growth factor and insulin-like growth factor 1.

OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.

Two-Compartment Perfusion MR IVIM Model to Investigate Normal and Pathological Placental Tissue.

BACKGROUND: Perfusion and diffusion coexist in the placenta and can be altered by pathologies. The two-perfusion model, where f1 and, f2 are the perfusion-fraction of the fastest and slowest perfusion compartment, respectively, and D is the diffusion coefficient, may help differentiate between normal and impaired placentas. PURPOSE: Investigate the potential of the two-perfusion IVIM model in differentiating between normal and abnormal placentas. STUDY-TYPE: Retrospective, case-control. POPULATION: 43 normal pregnancy, 9 fetal-growth-restriction (FGR), 6 small-for-gestational-age (SGA), 4 accreta, 1 increta and 2 percreta placentas. FIELD STRENGTH/SEQUENCE: Diffusion-weighted-echo planar imaging sequence at 1.5 T. ASSESSMENT: Voxel-wise signal-correction and fitting-controls were used to avoid overfitting obtaining that two-perfusion model fitted the observed data better than the IVIM model (Akaike weight: 0.94). The two-perfusion parametric-maps were quantified from ROIs in the fetal and maternal placenta and in the accretion zone of accreta placentas. The diffusion coefficient D was evaluated using a b ≥ 200 sec/mm2 -mono-exponential decay fit. IVIM metrics were quantified to fix f1 + f2 = fIVIM . STATISTICAL-TESTS: ANOVA with Dunn-Sidák's post-hoc correction and Cohen's d test were used to compare parameters between groups. Spearman's coefficient was evaluated to study the correlation between variables. A P-value<0.05 indicated a statistically significant difference. RESULTS: There was a significant difference in f1 between FGR and SGA, and significant differences in f2 and fIVIM between normal and FGR. The percreta + increta group showed the highest f1 values (Cohen's d = -2.66). The f2 between normal and percreta + increta groups showed Cohen's d = 1.12. Conversely, fIVIM had a small effective size (Cohen's d = 0.32). In the accretion zone, a significant correlation was found between f2 and GA (ρ = 0.90) whereas a significant negative correlation was found between fIVIM and D (ρ = -0.37 in fetal and ρ = -0.56 in maternal side) and f2 and D (ρ = -0.38 in fetal and ρ = -0.51 in maternal side) in normal placentas. CONCLUSION: The two-perfusion model provides complementary information to IVIM parameters that may be useful in identifying placenta impairment. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 1.

Reduction of Microvessel Number and Length in the Cerebellum of Purkinje Cell Degeneration Mice.

Degenerative effects of nerve tissues are often accompanied by changes in vascularization. In this regard, knowledge about hereditary cerebellar degeneration is limited. In this study, we compared the vascularity of the individual cerebellar components of 3-month-old wild-type mice (n = 8) and Purkinje cell degeneration (pcd) mutant mice, which represent a model of hereditary cerebellar degeneration (n = 8). Systematic random samples of tissue sections were processed, and laminin was immunostained to visualize microvessels. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in cerebellar layers. Our results in pcd mice revealed a 45% (p < 0.01) reduction in the total volume of the cerebellum, a 28% (p < 0.05) reduction in the total number of vessels and a lower total length, approaching 50% (p < 0.001), compared to the control mice. In pcd mutants, cerebellar degeneration is accompanied by significant reduction in the microvascular network that is proportional to the cerebellar volume reduction therefore does not change density of in the cerebellar gray matter of pcd mice.

Vasa vasorum interna in the carotid wall of active forms of Takayasu arteritis evidenced by ultrasound localization microscopy.

Introduction: Takayasu arteritis (TA) is associated with microvascularization of the wall of large arteries and is related to inflammation. Ultrasound localization microscopy (ULM), combining ultrafast ultrasound imaging with microbubble (MB) injection, can track the path of MBs within the arterial wall and thus provide imaging of the vasa vasorum. From the analysis of MB tracks in the common carotid arteries of patients with active TA, we report the presence of microvessels in connection with the carotid lumen (i.e., vasa vasorum interna [VVI]). Methods: ULM maps were obtained on five patients with active disease in the observational single-center series of the TAK-UF study. MB tracks connected to the carotid lumen were automatically identified, allowing the reconstruction of VVI. Results: MB tracking allows us to observe a microvascular network on the inner part of the wall, with some vessels in communication with the carotid lumen. This type of vessel was identified in all patients with active TA (n = 5) with a median of 2.2 [1.1-3.0] vessels per acquisition (2D longitudinal view of 3 cm of the common carotid artery). The blood flow within these vessels is mainly centrifugal; that is, toward the adventitia (88% [54-100] of MB tracks with flow directed to the outer part of the wall). Conclusion: VVI are present in humans in the case of active TA and emphasize the involvement of the intima in the pathological process. ClinicalTrials.gov Identifier: NCT03956394.

Deep Learning of the Retina Enables Phenome- and Genome-Wide Analyses of the Microvasculature.

BACKGROUND: The microvasculature, the smallest blood vessels in the body, has key roles in maintenance of organ health and tumorigenesis. The retinal fundus is a window for human in vivo noninvasive assessment of the microvasculature. Large-scale complementary machine learning-based assessment of the retinal vasculature with phenome-wide and genome-wide analyses may yield new insights into human health and disease. METHODS: We used 97 895 retinal fundus images from 54 813 UK Biobank participants. Using convolutional neural networks to segment the retinal microvasculature, we calculated vascular density and fractal dimension as a measure of vascular branching complexity. We associated these indices with 1866 incident International Classification of Diseases-based conditions (median 10-year follow-up) and 88 quantitative traits, adjusting for age, sex, smoking status, and ethnicity. RESULTS: Low retinal vascular fractal dimension and density were significantly associated with higher risks for incident mortality, hypertension, congestive heart failure, renal failure, type 2 diabetes, sleep apnea, anemia, and multiple ocular conditions, as well as corresponding quantitative traits. Genome-wide association of vascular fractal dimension and density identified 7 and 13 novel loci, respectively, that were enriched for pathways linked to angiogenesis (eg, vascular endothelial growth factor, platelet-derived growth factor receptor, angiopoietin, and WNT signaling pathways) and inflammation (eg, interleukin, cytokine signaling). CONCLUSIONS: Our results indicate that the retinal vasculature may serve as a biomarker for future cardiometabolic and ocular disease and provide insights into genes and biological pathways influencing microvascular indices. Moreover, such a framework highlights how deep learning of images can quantify an interpretable phenotype for integration with electronic health record, biomarker, and genetic data to inform risk prediction and risk modification.

BACE2 deficiency impairs expression and function of endothelial nitric oxide synthase in brain endothelial cells.

Beta-site amyloid precursor protein (APP)-cleaving enzyme 2 (BACE2) is highly expressed in cerebrovascular endothelium. Notably, BACE2 is one of the most downregulated genes in cerebrovascular endothelium derived from patients with Alzheimer's disease. The present study was designed to determine the role of BACE2 in control of expression and function of endothelial nitric oxide synthase (eNOS). Genetic downregulation of BACE2 with small interfering RNA (BACE2siRNA) in human brain microvascular endothelial cells (BMECs) significantly decreased expression of eNOS and elevated levels of eNOS phosphorylated at threonine residue Thr495, thus leading to reduced production of nitric oxide (NO). BACE2siRNA also suppressed expression of APP and decreased production and release of soluble APPα (sAPPα). In contrast, adenovirus-mediated overexpression of APP increased expression of eNOS. Consistent with these observations, nanomolar concentrations of sAPPα and APP 17mer peptide (derived from sAPPα) augmented eNOS expression. Further analysis established that γ-aminobutyric acid type B receptor subunit 1 and Krüppel-like factor 2 may function as downstream molecular targets significantly contributing to BACE2/APP/sAPPα-induced up-regulation of eNOS. In agreement with studies on cultured human endothelium, endothelium-dependent relaxations to acetylcholine and basal production of cyclic GMP were impaired in cerebral arteries of BACE2-deficient mice. We propose that in the brain blood vessels, BACE2 may function as a vascular protective protein.

Amyloid beta peptides (Aß) from Alzheimer's disease neuronal secretome induce endothelial activation in a human cerebral microvessel model.

In Alzheimer's disease (AD), secretion and deposition of amyloid beta peptides (Aß) have been associated with blood-brain barrier dysfunction. However, the role of Aß in endothelial cell (EC) dysfunction remains elusive. Here we investigated AD mediated EC activation by studying the effect of Aß secreted from human induced pluripotent stem cell-derived cortical neurons (hiPSC-CN) harboring a familial AD mutation (Swe+/+) on human brain microvascular endothelial cells (HBMECs) in 2D and 3D perfusable microvessels. We demonstrated that increased Aß levels in Swe+/+ conditioned media (CM) led to stress fiber formation and upregulation of genes associated with endothelial inflammation and immune-adhesion. Perfusion of Aß-rich Swe+/+ CM induced acute formation of von Willebrand factor (VWF) fibers in the vessel lumen, which was attenuated by reducing Aß levels in CM. Our findings suggest that Aß peptides can trigger rapid inflammatory and thrombogenic responses within cerebral microvessels, which may exacerbate AD pathology.

Diabetic microvascular disease in non-classical beds: the hidden impact beyond the retina, the kidney, and the peripheral nerves.

Diabetes microangiopathy, a hallmark complication of diabetes, is characterised by structural and functional abnormalities within the intricate network of microvessels beyond well-known and documented target organs, i.e., the retina, kidney, and peripheral nerves. Indeed, an intact microvascular bed is crucial for preserving each organ's specific functions and achieving physiological balance to meet their respective metabolic demands. Therefore, diabetes-related microvascular dysfunction leads to widespread multiorgan consequences in still-overlooked non-traditional target organs such as the brain, the lung, the bone tissue, the skin, the arterial wall, the heart, or the musculoskeletal system. All these organs are vulnerable to the physiopathological mechanisms that cause microvascular damage in diabetes (i.e., hyperglycaemia-induced oxidative stress, inflammation, and endothelial dysfunction) and collectively contribute to abnormalities in the microvessels' structure and function, compromising blood flow and tissue perfusion. However, the microcirculatory networks differ between organs due to variations in haemodynamic, vascular architecture, and affected cells, resulting in a spectrum of clinical presentations. The aim of this review is to focus on the multifaceted nature of microvascular impairment in diabetes through available evidence of specific consequences in often overlooked organs. A better understanding of diabetes microangiopathy in non-target organs provides a broader perspective on the systemic nature of the disease, underscoring the importance of recognising the comprehensive range of complications beyond the classic target sites.

Development of angiogenic periglomerular microvessels after acute glomerular lesions in IgA nephropathy.

AIM: To clarify the clinicopathological characteristics and role of periglomerular angiogenesis in IgA nephropathy. METHODS AND RESULTS: The renal biopsy specimens of 114 patients with IgA nephropathy were examined. Among them, 46 (40%) showed periglomerular angiogenesis around the glomeruli. CD34 and α-smooth muscle actin (α-SMA) staining in serial sections revealed that these vessels contained CD34+ α-SMA+ microarterioles along with CD34+ α-SMA- capillaries. We termed these "periglomerular microvessels (PGMVs)". Patients with PGMVs (PGMV group) had clinically and histologically more severe disease than those without PGMVs (non-PGMV group) at the time of biopsy. Even after adjusting for age, there were significant differences in the degree of proteinuria and estimated glomerular filtration rate reduction between the PGMV and non-PGMV groups. The PGMV group showed a higher incidence of segmental and global glomerulosclerosis and crescentic lesions than the non-PGMV group (P < 0.01). Here, PGMVs were undetectable in the acute and active inflammation phase, but were observed in the acute to chronic or chronic glomerular remodelling phase. PGMVs mainly developed around glomerular adherent lesions to the Bowman's capsule with small or minimal glomerular sclerotic lesions. Conversely, they were rarely observed in segmental sclerosis areas. CONCLUSION: The PGMV group is clinically and pathologically more severe than the non-PGMV group; however, they were undetectable in segmental sclerosis with mesangial matrix accumulation. PGMVs might occur after acute/active glomerular lesions, suggesting that PGMVs may inhibit segmental glomerulosclerosis progression and could be a marker for good repair response after acute/active glomerular injury in severe IgA nephropathy cases.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies.

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

New Directions in Therapeutic Angiogenesis and Arteriogenesis in Peripheral Arterial Disease.

The prevalence of peripheral arterial disease (PAD) in the United States exceeds 10 million people, and PAD is a significant cause of morbidity and mortality across the globe. PAD is typically caused by atherosclerotic obstructions in the large arteries to the leg(s). The most common clinical consequences of PAD include pain on walking (claudication), impaired functional capacity, pain at rest, and loss of tissue integrity in the distal limbs that may lead to lower extremity amputation. Patients with PAD also have higher than expected rates of myocardial infarction, stroke, and cardiovascular death. Despite advances in surgical and endovascular procedures, revascularization procedures may be suboptimal in relieving symptoms, and some patients with PAD cannot be treated because of comorbid conditions. In some cases, relieving obstructive disease in the large conduit arteries does not assure complete limb salvage because of severe microvascular disease. Despite several decades of investigational efforts, medical therapies to improve perfusion to the distal limb are of limited benefit. Whereas recent studies of anticoagulant (eg, rivaroxaban) and intensive lipid lowering (such as PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitors) have reduced major cardiovascular and limb events in PAD populations, chronic ischemia of the limb remains largely resistant to medical therapy. Experimental approaches to improve limb outcomes have included the administration of angiogenic cytokines (either as recombinant protein or as gene therapy) as well as cell therapy. Although early angiogenesis and cell therapy studies were promising, these studies lacked sufficient control groups and larger randomized clinical trials have yet to achieve significant benefit. This review will focus on what has been learned to advance medical revascularization for PAD and how that information might lead to novel approaches for therapeutic angiogenesis and arteriogenesis for PAD.

Optimal heat transport induced by magnetic nanoparticle delivery in vascularised tumours.

We describe a novel mathematical model for blood flow, delivery of nanoparticles, and heat transport in vascularised tumour tissue. The model, which is derived via the asymptotic homogenisation technique, provides a link between the macroscale behaviour of the system and its underlying, tortuous micro-structure, as parametrised in Penta and Ambrosi (2015). It consists of a double Darcy's law, coupled with a double advection-diffusion-reaction system describing heat transport, and an advection-diffusion-reaction equation for transport and adhesion of particles. Particles are assumed sufficiently large and do not extravasate to the tumour interstitial space but blood and heat can be exchanged between the two compartments. Numerical simulations of the model are performed using a finite element method to investigate cancer hyperthermia induced by the application of magnetic field applied to injected iron oxide nanoparticles. Since tumour microvasculature is more tortuous than that of healthy tissue and thus suboptimal in terms of fluid and drug transport, we study the influence of the vessels' geometry on tumour temperature. Effective and safe hyperthermia treatment requires tumour temperature within certain target range, generally estimated between 42 °C and 46 °C, for a certain target duration, typically 0.5h to 2h. As temperature is difficult to measure in situ, we use our model to determine the ranges of tortuosity of the microvessels, magnetic intensity, injection time, wall shear stress rate, and concentration of nanoparticles required to achieve given target conditions.