Intimal Macrovascular Pericytes: Their Role in Vascular Biology and Atherogenesis.

Atherosclerosis remains a major challenge to global healthcare despite decades of research and constant trials of novel therapeutic approaches. One feature that makes atherosclerosis treatment so elusive is an insufficient understanding of its origins and the early stages of the pathological process, which limits our means of effective prevention of the disease. Macrovascular pericytes are cells with distinct shapes that are located in the arterial wall of larger vessels and are in many aspects similar to microvascular pericytes that maintain the functionality of small vessels and capillaries. This cell type combines the residual contractile function of smooth muscle cells with a distinct stellar shape that allows these cells to make numerous contacts between themselves and the adjacent endothelial layer. Moreover, pericytes can take part in the immune defense and are able to take up lipids in the course of atherosclerotic lesion development. In growing atherosclerotic plaques, the morphology and function of pericytes change dramatically due to phagocytic and synthetic phenotypes that are actively involved in lipid accumulation and extracellular matrix synthesis. In this review, we summarize our knowledge of this less-studied cell type and its role in atherosclerosis.

Unveiling the Mysteries of the Blood-Brain Barrier: The Problem of the Brain/spinal Pharmacotherapy.

The most critical issue impeding the development of innovative cerebrospinal medications is the blood-brain barrier (BBB). The BBB limits the ability of most medications to penetrate the brain to the CNS. The BBB structure and functions are summarized, with the physical barrier generated by endothelial tight junctions and the transport barrier formed by transporters within the membrane and vesicular processes. The functions of connected cells, particularly the end feet of astrocytic glial cells, microglia, and pericytes, are described. The drugs that cross the blood brain barrier are explained below along with their mechanisms. Some of the associated conditions and problems are given.

Contrast-enhanced ultrasound imaging detects anatomical and functional changes in rat cervical spine microvasculature with normal aging.

Normal aging is associated with significant deleterious cerebrovascular changes; these have been implicated in disease pathogenesis and increased susceptibility to ischemic injury. While these changes are well documented in the brain, few studies have been conducted in the spinal cord. Here, we utilize specialized contrast-enhanced ultrasound (CEUS) imaging to investigate age-related changes in cervical spinal vascular anatomy and hemodynamics in male Fisher 344 rats, a common strain in aging research. Aged rats (24-26 mo., N=6) exhibited significant tortuosity in the anterior spinal artery and elevated vascular resistance compared to adults (4-6 mo., N=6; tortuosity index 2.20±0.15 vs 4.74±0.45, p<0.05). Baseline blood volume was lower in both larger vessels and the microcirculation in the aged cohort, specifically in white matter (4.44e14±1.37e13 vs 3.66e14±2.64e13 CEUS bolus AUC, p<0.05). To elucidate functional differences, animals were exposed to a hypoxia challenge; whereas adult rats exhibited significant functional hyperemia in both gray and white matter (GM: 1.13±0.10-fold change from normoxia, p<0.05; WM: 1.16±0.13, p<0.05), aged rats showed no response. Immunohistochemistry revealed reduced pericyte coverage and activated microglia behavior in aged rats, which may partially explain the lack of vascular response. This study provides the first in vivo description of age-related hemodynamic differences in the cervical spinal cord.

Comprehensive identification, isolation, and culture of human breast cell types.

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing-and a thorough dissection of the genes and pathways defining each cell type. Whereas the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the twelve major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind- and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

Nerve-Glial antigen 2: unmasking the enigmatic cellular identity in the central nervous system.

Chondroitin sulfate proteoglycans (CSPGs) are fundamental components of the extracellular matrix in the central nervous system (CNS). Among these, the Nerve-Glial antigen 2 (NG2) stands out as a transmembrane CSPG exclusively expressed in a different population of cells collectively termed NG2-expressing cells. These enigmatic cells, found throughout the developing and adult CNS, have been indicated with various names, including NG2 progenitor cells, polydendrocytes, synantocytes, NG2 cells, and NG2-Glia, but are more commonly referred to as oligodendrocyte progenitor cells. Characterized by high proliferation rates and unique morphology, NG2-expressing cells stand apart from neurons, astrocytes, and oligodendrocytes. Intriguingly, some NG2-expressing cells form functional glutamatergic synapses with neurons, challenging the long-held belief that only neurons possess the intricate machinery required for neurotransmission. In the CNS, the complexity surrounding NG2-expressing cells extends to their classification. Additionally, NG2 expression has been documented in pericytes and immune cells, suggesting a role in regulating brain innate immunity and neuro-immune crosstalk in homeostasis. Ongoing debates revolve around their heterogeneity, potential as progenitors for various cell types, responses to neuroinflammation, and the role of NG2. Therefore, this review aims to shed light on the enigma of NG2-expressing cells by delving into their structure, functions, and signaling pathways. We will critically evaluate the literature on NG2 expression across the CNS, and address the contentious issues surrounding their classification and roles in neuroinflammation and neurodegeneration. By unraveling the intricacies of NG2-expressing cells, we hope to pave the way for a more comprehensive understanding of their contributions to CNS health and during neurological disorders.

25-Hydroxycholesterol attenuates tumor necrosis factor alpha-induced blood-brain barrier breakdown in vitro.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses.

Cell isolation protocols from brain tissue include prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Particularly for microglia and vascular cells, current isolation methods produce lower yields, necessitating addition of an enrichment step, and use of large tissue volumes - in most cases whole brain tissue - to obtain sufficient yields. Here, we developed a simple, rapid, and reproducible cell isolation method for generating single-cell suspensions from micro-dissected brain regions, enriched for microglia and vascular cells, without an enrichment step. Cells isolated using this method are suitable for molecular profiling studies using 10 × Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.

Breaking New Ground: Unraveling Sinonasal Hemangiopericytoma Through a Rare Case Series on Endoscopic Excision and Postoperative Monitoring.

Hemangiopericytoma is a rare vascular neoplasm derived from pericytes, with uncertain malignant potential. It mainly occurs in the retroperitoneum and lower extremities, with a lower incidence in the head and neck region and nasal cavity. Diagnosis is aided by histopathological examination and immunohistochemistry. Surgical excision is the preferred treatment, with different approaches depending on tumour size. Endoscopic excision is suitable for small tumours, while larger ones may require external approaches. The recurrence rate is approximately 25%, emphasizing the importance of long-term follow-up. Our study aims to discuss a rare series of sinonasal hemangiopericytoma cases, their clinical presentation, and their management. In this study, we are discussing the prospective study of six cases of sinonasal hemangiopericytoma that were presented to a tertiary hospital, from June 2017 to June 2023, with complaints of nasal obstruction and bleeding episodes. They were assessed with a detailed history, blood investigations, radiological investigations, and diagnostic nasal examination, and underwent endoscopic surgical excision of the nasal mass, with the diagnosis confirmed by histopathological examination and immunohistochemistry. All cases were followed up for 1 year postoperatively, except one case which missed follow-up after 6 months and with no postoperative complications and recurrences. All six cases came with complaints of nasal obstruction and bleeding from the nasal cavity. All six cases underwent endoscopic surgical excision of the tumour and were followed for 1 year in five cases; one case missed follow-up after 6 months of postoperative follow-up, but no recurrence was noted in all the cases. For small-sized hemangiopericytoma tumours, endoscopic excision offers benefits such as improved visualization, easy resection, preservation of the normal anatomical structure, and maintenance of physiological function in the sinonasal cavities. With a recurrence rate of approximately 25%, surgical excision and long-term follow-up play essential roles in successful tumour management.

Endothelin-1 (ET-1) contributes to senescence and phenotypic changes in brain pericytes in diabetes-mimicking conditions.

Diabetes mediates endothelial dysfunction and increases the risk of Alzheimer's disease and related dementias. Diabetes also dysregulates the ET system. ET-1-mediated constriction of brain microvascular pericytes (BMVPCs) has been shown to contribute to brain hypoperfusion. Cellular senescence, a process that arrests the proliferation of harmful cells and instigates phenotypical changes and proinflammatory responses in endothelial cells that impact their survival and function. Thus, we hypothesized that ET-1 mediates BMVPC senescence and phenotypical changes in diabetes-like conditions. Human BMVPCs were incubated in diabetes-like conditions with or without ET-1 (1 µmol/L) for 3 and 7 days. Hydrogen peroxide (100 µmol/L H2O2) was used as a positive control for senescence and to mimic ischemic conditions. Cells were stained for senescence-associated ß-galactosidase or processed for immunoblotting and quantitative real-time PCR analyses. In additional experiments, cells were stimulated with ET-1 in the presence or absence of ETA receptor antagonist BQ-123 (20 µmol/L) or ETB receptor antagonist BQ-788 (20 µmol/L). ET-1 stimulation increased ß-galactosidase accumulation which was prevented by BQ-123. ET-1 also increased traditional senescence marker p16 protein and pericyte-specific senescence markers, TGFB1i1, PP1CA, and IGFBP7. Furthermore, ET-1 stimulated contractile protein α-SMA and microglial marker ostepontin in high glucose suggesting a shift toward an ensheathing or microglia-like phenotype. In conclusion, ET-1 triggers senescence, alters ETA and ETB receptors, and causes phenotypical changes in BMVPCs under diabetes-like conditions. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of pericyte senescence and phenotypical changes in VCID.

Loss of TGFß-Mediated Repression of Angiopoietin-2 in Pericytes Underlies Germinal Matrix Hemorrhage Pathogenesis.

BACKGROUND: TGF (transforming growth factor)-ß pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFß receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. METHODS: Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. RESULTS: Herein, we establish that the TGFß/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFß-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFß-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. CONCLUSIONS: This study demonstrates that TGFß-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.

Single-cell dissection of the human blood-brain barrier and glioma blood-tumor barrier.

The blood-brain barrier (BBB) serves as a crucial vascular specialization, shielding and nourishing brain neurons and glia while impeding drug delivery. Here, we conducted single-cell mRNA sequencing of human cerebrovascular cells from 13 surgically resected glioma samples and adjacent normal brain tissue. The transcriptomes of 103,230 cells were mapped, including 57,324 endothelial cells (ECs) and 27,703 mural cells (MCs). Both EC and MC transcriptomes originating from lower-grade glioma were indistinguishable from those of normal brain tissue, whereas transcriptomes from glioblastoma (GBM) displayed a range of abnormalities. Among these, we identified LOXL2-dependent collagen modification as a common GBM-dependent trait and demonstrated that inhibiting LOXL2 enhanced chemotherapy efficacy in both murine and human patient-derived xenograft (PDX) GBM models. Our comprehensive single-cell RNA sequencing-based molecular atlas of the human BBB, coupled with insights into its perturbations in GBM, holds promise for guiding future investigations into brain health, pathology, and therapeutic strategies.

Hyperglycemia-induced oxidative stress exacerbates mitochondrial apoptosis damage to cochlear stria vascularis pericytes via the ROS-mediated Bcl-2/CytC/AIF pathway.

OBJECTIVES: Diabetes is closely linked to hearing loss, yet the exact mechanisms remain unclear. Cochlear stria vascularis and pericytes (PCs) are crucial for hearing. This study investigates whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes via elevated ROS levels due to oxidative stress, impacting hearing loss. METHODS: We established a type II diabetes model in C57BL/6J mice and used auditory brainstem response (ABR), Evans blue staining, HE staining, immunohistochemistry, and immunofluorescence to observe changes in hearing, blood-labyrinth barrier (BLB) permeability, stria vascularis morphology, and apoptosis protein expression. Primary cultured stria vascularis pericytes were subjected to high glucose, and apoptosis levels were assessed using flow cytometry, Annexin V-FITC, Hoechst 33342 staining, Western blot, Mitosox, and JC-1 probes. RESULTS: Diabetic mice showed decreased hearing thresholds, reduced stria vascularis density, increased oxidative stress, cell apoptosis, and decreased antioxidant levels. High glucose exposure increased apoptosis and ROS content in pericytes, while mitochondrial membrane potential decreased, with AIF and cytochrome C (CytC) released from mitochondria to the cytoplasm. Adding oxidative scavengers reduced AIF and CytC release, decreasing pericyte apoptosis. DISCUSSION: Hyperglycemia may induce mitochondrial apoptosis of cochlear stria vascularis pericytes through oxidative stress.

Pericytes: jack-of-all-trades in cancer-related inflammation.

Pericytes, recognized as mural cells, have long been described as components involved in blood vessel formation, playing a mere supporting role for endothelial cells (ECs). Emerging evidence strongly suggests their multifaceted roles in tissues and organs. Indeed, pericytes exhibit a remarkable ability to anticipate endothelial cell behavior and adapt their functions based on the specific cells they interact with. Pericytes can be activated by pro-inflammatory stimuli and crosstalk with immune cells, actively participating in their transmigration into blood vessels. Moreover, they can influence the immune response, often sustaining an immunosuppressive phenotype in most of the cancer types studied. In this review, we concentrate on the intricate crosstalk between pericytes and immune cells in cancer, highlighting the primary evidence regarding pericyte involvement in primary tumor mass dynamics, their contributions to tumor reprogramming for invasion and migration of malignant cells, and their role in the formation of pre-metastatic niches. Finally, we explored recent and emerging pharmacological approaches aimed at vascular normalization, including novel strategies to enhance the efficacy of immunotherapy through combined use with anti-angiogenic drugs.

Role of blood-brain barrier dysfunction in the development of poststroke epilepsy.

Stroke is a major contributor to mortality and morbidity worldwide and the most common cause of epilepsy in the elderly in high income nations. In recent years, it has become increasingly evident that both ischemic and hemorrhagic strokes induce dysfunction of the blood-brain barrier (BBB), and that this impairment can contribute to epileptogenesis. Nevertheless, studies directly comparing BBB dysfunction and poststroke epilepsy (PSE) are largely absent. Therefore, this review summarizes the role of BBB dysfunction in the development of PSE in animal models and clinical studies. There are multiple mechanisms whereby stroke induces BBB dysfunction, including increased transcytosis, tight junction dysfunction, spreading depolarizations, astrocyte and pericyte loss, reactive astrocytosis, angiogenesis, matrix metalloproteinase activation, neuroinflammation, adenosine triphosphate depletion, oxidative stress, and finally cell death. The degree to which these effects occur is dependent on the severity of the ischemia, whereby cell death is a more prominent mechanism of BBB disruption in regions of critical ischemia. BBB dysfunction can contribute to epileptogenesis by increasing the risk of hemorrhagic transformation, increasing stroke size and the amount of cerebral vasogenic edema, extravasation of excitatory compounds, and increasing neuroinflammation. Furthermore, albumin extravasation after BBB dysfunction contributes to epileptogenesis primarily via increased transforming growth factor ß signaling. Finally, seizures themselves induce BBB dysfunction, thereby contributing to epileptogenesis in a cyclical manner. In repairing this BBB dysfunction, pericyte migration via platelet-derived growth factor ß signaling is indispensable and required for reconstruction of the BBB, whereby astrocytes also play a role. Although animal stroke models have their limitations, they provide valuable insights into the development of potential therapeutics designed to restore the BBB after stroke, with the ultimate goal of improving outcomes and minimizing the occurrence of PSE. In pursuit of this goal, rapamycin, statins, losartan, semaglutide, and metformin show promise, whereby modulation of pericyte migration could also be beneficial.

Macrophages play a crucial role in vascular smooth muscle cell coverage.

The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFß-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively-active Foxo1-overexpression, substantial accumulation of TGFß1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFß-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in an excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.

Robust CXCL10/IP-10 and CCL5/RANTES Production Induced by Tick-Borne Encephalitis Virus in Human Brain Pericytes Despite Weak Infection.

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.

Endothelial Cell-Specific Prolyl Hydroxylase-2 Deficiency Augments Angiotensin II-Induced Arterial Stiffness and Cardiac Pericyte Recruitment in Mice.

BACKGROUND: Endothelial prolyl hydroxylase-2 (PHD2) is essential for pulmonary remodeling and hypertension. In the present study, we investigated the role of endothelial PHD2 in angiotensin II-mediated arterial stiffness, pericyte recruitment, and cardiac fibrosis. METHODS AND RESULTS: Chondroitin sulfate proteoglycan 4 tracing reporter chondroitin sulfate proteoglycan 4- red fluorescent protein (DsRed) transgenic mice were crossed with PHD2flox/flox (PHD2f/f) mice and endothelial-specific knockout of PHD2 (PHD2ECKO) mice. Transgenic PHD2f/f (TgPHD2f/f) mice and TgPHD2ECKO mice were infused with angiotensin II for 4 weeks. Arterial thickness, stiffness, and histological and immunofluorescence of pericytes and fibrosis were measured. Infusion of TgPHD2f/f mice with angiotensin II resulted in a time-dependent increase in pulse-wave velocity. Angiotensin II-induced pulse-wave velocity was further elevated in the TgPHD2ECKO mice. TgPHD2ECKO also reduced coronary flow reserve compared with TgPHD2f/f mice infused with angiotensin II. Mechanistically, knockout of endothelial PHD2 promoted aortic arginase activity and angiotensin II-induced aortic thickness together with increased transforming growth factor-ß1 and ICAM-1/VCAM-1 expression in coronary arteries. TgPHD2f/f mice infused with angiotensin II for 4 weeks exhibited a significant increase in cardiac fibrosis and hypertrophy, which was further developed in the TgPHD2ECKO mice. Chondroitin sulfate proteoglycan 4 pericyte was traced by DsRed+ staining and angiotensin II infusion displayed a significant increase of DsRed+ pericytes in the heart, as well as a deficiency of endothelial PHD2, which further promoted angiotensin II-induced pericyte increase. DsRed+ pericytes were costained with fibroblast-specific protein 1 and α-smooth muscle actin for measuring pericyte-myofibroblast cell transition. The knockout of endothelial PHD2 increased the amount of DsRed+/fibroblast-specific protein 1+ and DsRed+/α-smooth muscle actin+ cells induced by angiotensin II infusion. CONCLUSIONS: Knockout of endothelial PHD2 enhanced angiotensin II-induced cardiac fibrosis by mechanisms involving increasing arterial stiffness and pericyte-myofibroblast cell transitions.

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

BACKGROUND: It has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure. METHOD: Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC-MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter. RESULTS: Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells. CONCLUSION: We reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

Selective mural cell recruitment of pericytes to networks of assembling endothelial cell-lined tubes.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

Isolation methods and characterization of primary rat neurovascular cells.

BACKGROUND: There is significant interest in isolating cells of the blood-brain barrier (BBB) for use in in vitro screening of therapeutics and analyzing cell specific roles in neurovascular pathology. Primary brain cells play an advantageous role in BBB models; however, isolation procedures often do not produce cells at high enough yields for experiments. In addition, although numerous reports provide primary cell isolation methods, the field is lacking in documentation and detail of expected morphological changes that occur throughout culturing and there are minimal troubleshooting resources. Here, we present simplified, robust, and reproducible methodology for isolating astrocytes, pericytes, and endothelial cells, and demonstrate several morphological benchmarks for each cell type throughout the process and culture timeframe. We also analyze common considerations for developing neurovascular cell isolation procedures and recommend solutions for troubleshooting. RESULTS: The presented methodology isolated astrocytes, pericytes, and endothelial cells and enabled cell attachment, maturation, and cell viability. We characterized milestones in cell maturation over 12 days in culture, a common timeline for applications of these cell types in BBB models. Phase contrast microscopy was used to show initial cell plating, attachment, and daily growth of isolated cells. Confocal microscopy images were analyzed to determine the identity of cell types and changes to cell morphology. Nuclear staining was also used to show the viability and proliferation of glial cells at four time points. Astrocyte branches became numerous and complex with increased culture time. Microglia, oligodendrocytes, and neurons were present in mixed glial cultures for 12 days, though the percentage of microglia and neurons expectedly decreased after passaging, with microglia demonstrating a less branched morphology. CONCLUSIONS: Neurovascular cells can be isolated through our optimized protocols that minimize cell loss and encourage the adhesion and proliferation of isolated cells. By identifying timepoints of viable glia and neurons within an astrocyte-dominant mixed culture, these cells can be used to evaluate drug targeting, uptake studies, and response to pathological stimulus in the neurovascular unit.