Vascular Stability Research Articles

Brain Microvascular Pericytes in Vascular Cognitive Impairment and Dementia.

Pericytes are unique, multi-functional mural cells localized at the abluminal side of the perivascular space in microvessels. Originally discovered in 19th century, pericytes had drawn less attention until decades ago mainly due to lack of specific markers. Recently, however, a growing body of evidence has revealed that pericytes play various important roles: development and maintenance of blood–brain barrier (BBB), regulation of the neurovascular system (e.g., vascular stability, vessel formation, cerebral blood flow, etc.), trafficking of inflammatory cells, clearance of toxic waste products from the brain, and acquisition of stem cell-like properties. In the neurovascular unit, pericytes perform these functions through coordinated crosstalk with neighboring cells including endothelial, glial, and neuronal cells. Dysfunction of pericytes contribute to a wide variety of diseases that lead to cognitive impairments such as cerebral small vessel disease (SVD), acute stroke, Alzheimer’s disease (AD), and other neurological disorders. For instance, in SVDs, pericyte degeneration leads to microvessel instability and demyelination while in stroke, pericyte constriction after ischemia causes a no-reflow phenomenon in brain capillaries. In AD, which shares some common risk factors with vascular dementia, reduction in pericyte coverage and subsequent microvascular impairments are observed in association with white matter attenuation and contribute to impaired cognition. Pericyte loss causes BBB-breakdown, which stagnates amyloid β clearance and the leakage of neurotoxic molecules into the brain parenchyma. In this review, we first summarize the characteristics of brain microvessel pericytes, and their roles in the central nervous system. Then, we focus on how dysfunctional pericytes contribute to the pathogenesis of vascular cognitive impairment including cerebral ‘small vessel’ and ‘large vessel’ diseases, as well as AD. Finally, we discuss therapeutic implications for these disorders by targeting pericytes.

CD151 Alleviates Early Blood-Brain Barrier Dysfunction After Experimental Focal Brain Ischemia in Rats.

Preservation of the blood-brain barrier (BBB) function is a potential protective strategy against cerebral ischemic injuries. CD151 has a beneficial effect in maintaining vascular stability and plays a role in pro-angiogenesis. Both vascular stability and angiogenesis can affect BBB function. Therefore, we aimed to examine the action of CD151 in regulating BBB permeability after cerebral ischemic injury in the present study. Using a transient focal cerebral ischemia (tFCI) rat model, we established that CD151 overexpression in the brain mitigated the leakage of endogenous IgG at 6-24h after tFCI in vivo. Moreover, we found that CD151 can decrease the diffusion of macromolecules through monolayer brain microvessel endothelial cells (BMVECs) after glucose and oxygen deprivation (OGD)-reoxygenation in vitro. Furthermore, overexpression of CD151 in BMVECs suppressed OGD-reoxygenation-induced F-actin formation and RhoA activity. However, while preserving BBB integrity after tFCI, CD151 overexpression did not affect the post-stroke outcomes. Taken together, the present study demonstrated that CD151 overexpression in the brain protects BBB permeability at early phase after tFCI. CD151 may be a potential target for early BBB protection in ischemic stroke.

Developing a FRET‐based Platform of Monitoring GPCR‐induced p38 Activation Illuminates the Foundational Basis of Spatiotemporal Regulation in Proinflammatory Signaling

Mitogen‐activated protein kinase (MAPK) p38 is a critical component in pro‐inflammatory signaling associated with chronic obstructive pulmonary disorder (COPD) and asthma, and is considered a potential therapeutic target for proinflammatory disease. Unfortunately, direct targeting of p38 activity has proven unsuccessful in modulating inflammation, due to the physiologically ubiquitous necessity of p38 activity in tissues. G‐protein coupled receptor (GPCR) stimulation has been shown to mediate p38 autophosphorylation via interaction with TGF‐Beta Activated Kinase 1 Binding Protein 1 (TAB1) independently of typical p38 activation by MKK3/6. We predict that inhibition of p38‐TAB1 interaction will reduce pathological pro‐inflammatory signaling without affecting critical physiological p38 activation. We predicted that the spatiotemporal regulation of this under‐characterized pathway of p38 signaling is critical for downstream signaling responses associated with proinflammatory disease. In this work we have developed a Forster’s Resonance Energy Transfer (FRET) system of subcellular localized biosensors capable of detecting P38 activity. Through exploration of two GPCRs known to activate atypical p38 signaling (PAR1 and EP2) we demonstrate differential signaling kinetics of cytosolic and plasma membrane p38 activation in HeLa cells co‐transfected with the receptor and subcellular‐directed biosensor. The variances in subcellular p38 activation are the subject of current investigations. We have established a basis for further expansion of this system to include acute detection of nuclear and endosomal p38 activation, and will adapt this platform for small scale screening of p38 inhibitor compounds in stable cell lines expressing subcellular localized biosensors. Combined, these studies provide key foundational insight into the complex interplay and relationships between GPCRs and downstream effectors on vascular stability, inflammation, and angiogenesis, potentially leading to the discovery of novel therapeutic approaches to addressing chronic inflammatory disease.

Characterization of p38 Inflammatory Response in Human Pericytes

G protein‐coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro‐inflammation and pro‐angiogenic responses during chronic lung disease. Our studies have revealed an atypical (non‐canonical) pathway for p38 activation that induces auto‐phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein‐1 (TAB1) in endothelial cells. However, during angiogenesis, endothelial cells are known for their rapid reproduction, migration, and tubule formation. Pericyte cells serve to guide these endothelial cells, providing vascular stability and cytokine signaling. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells, but is hypothesized to play a key role in regulating G protein‐coupled receptor‐mediated control of vascular function. Very little is known about the cellular mechanisms that control atypical p38 activation, representing a novel therapeutic target. Our preliminary data shows that thrombin and histamine can activate p38 via the atypical p38 signaling pathway in primary human pericytes. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co‐cultured model of pericyte and endothelial cells. Using this model, we are the first to define the role of atypical p38 signaling in pericytes, along with their vascular formation and function. These studies represent a novel approach to regulate vascular p38 activity, pro‐inflammatory signaling, and chronic lung disease, and this model will provide us with the tools necessary to identify the role of p38 induced inflammation in the migration of pericyte cells.

Novel angiogenesis strategy to ameliorate pulmonary hypertension

ObjectiveTo select a suitable combination of classic angiogenic and vascular stabilization factors to improve the proliferation and maturity of neovascularization of lung tissue in a rat pulmonary arterial hypertension (PAH) model. MethodsPAH rat model was established by intraperitoneal injection of monocrotaline. Proangiogenic factors hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), as well as vascular stabilization factors angiopoietin-1 (Ang-1), platelet-derived growth factor, and transforming growth factor-beta were transfected by pairs into the lung tissue of rats with PAH through lentivirus. Four weeks later, pulmonary artery angiography and hemodynamic parameters were determined to testify the remission of PAH. Immunofluorescence staining and Western blot were performed to investigate the structure and function of neovascularization. ResultsThe pulmonary artery pressure and weight index of the right ventricle in HGF+Ang-1 and VEGF+Ang-1 groups were significantly decreased compared with vehicle group. The contrast medium filling time and right pulmonary artery root diameter were also significantly decreased. In addition, the maturity and perfusion of neovascularization in HGF+Ang-1 and VEGF+Ang-1 groups were promoted compared to vehicle group, and vascular leakage was reduced. Finally, the adherens junction integrity of vascular endothelial cells in HGF+Ang-1 and VEGF+Ang-1 combinations was upregulated compared with other combinations. ConclusionsHGF+Ang-1 transfection and VEGF+Ang-1 transfection alleviate PAH by promoting maturation and stability of new blood vessels, which may be potential candidates for PAH treatment.

Collagen Microfibers Induce Blood Capillary Orientation and Open Vascular Lumen.

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.

Investigation of the Possible Role of Tie2 Pathway and TEK Gene in Asthma and Allergic Conjunctivitis.

Tie2, coded by the TEK gene, is a tyrosine kinase receptor and plays a central role in vascular stability. It was suggested that variations in the TEK gene might influence the susceptibility to asthma and allergic conjunctivitis. The aim of this study was to further investigate these suggestions, involving different populations and to study the Tie2 related pathway on a mouse model of asthma. The discovery, stage I cohort involved 306 patients with moderate and severe allergic rhinitis, the stage II study consisted of four cohorts, namely, adult and pediatric asthmatics and corresponding controls. Altogether, there were 1,258 unrelated individuals in these cohorts, out of which 63.9% were children and 36.1% were adults. In stage I, 112 SNPs were screened in the TEK gene of the patients in order to search for associations with asthma and allergic conjunctivitis. The top associated SNPs were selected for association studies on the replication cohorts. The rs3824410 SNP was nominally associated with a reduced risk of asthma in the stage I cohort and with severe asthma within the asthmatic population (p=0.009; OR=0.48) in the replication cohort. In the stage I study, 5 SNPs were selected in conjunctivitis. Due to the low number of adult patients with conjunctivitis, only children were involved in stage II. Within the asthmatic children, the rs622232 SNP was associated with conjunctivitis in boys in the dominant model (p=0.004; OR=4.76), while the rs7034505 showed association to conjunctivitis in girls (p=0.012; OR=2.42). In the lung of a mouse model of asthma, expression changes of 10 Tie2 pathway-related genes were evaluated at three points in time. Eighty percent of the selected genes showed significant changes in their expressions at least at one time point during the process, leading from sensitization to allergic airway inflammation. The expressions of both the Tek gene and its ligands showed a reduced level at all time points. In conclusion, our results provide additional proof that the Tie2 pathway, the TEK gene and its variations might have a role in asthma and allergic conjunctivitis. The gene and its associated pathways can be potential therapeutic targets in both diseases.

Longitudinal neural and vascular structural dynamics produced by chronic microelectrode implantation

Implanted microelectrode arrays sense local neuronal activity, signals which are used as control commands for brain computer interface (BCI) technology. Patients with tetraplegia have used BCI technology to achieve an extraordinary degree of interaction with their local environment. However, current microelectrode arrays for BCIs lose the ability to record high-quality neural signals in the months-to-years following implantation. Very little is known regarding the dynamic response of neurons and vasculature in the months following electrode array implantation, but loss of structural integrity near the electrode may contribute to the degradation of recording signals. Here, we use in-vivo dual-modality imaging to characterize neuronal and vasculature structures in the same animal for 3 months following electrode insertion. We find ongoing neuronal atrophy, but relative vascular stability, in close proximity to the electrode, along with evidence suggesting links between rare, abrupt hypoxic events and neuronal process atrophy.

Exogenous bone marrow derived-putative endothelial progenitor cells attenuate ischemia reperfusion-induced vascular injury and renal fibrosis in mice dependent on pericytes.

Rationale: Capillaries are composed of endothelial cells and the surrounding mural cells, pericytes. Microvascular repair after injury involves not only the proliferation of endothelial cells but also pericyte-based vessel stabilization. Exogenous bone marrow derived-putative endothelial progenitor cells (b-pEPCs) have the potential for vascular repair; however, their effect on vascular structure stabilization and pericyte-related pathobiological outcomes in the injured kidney has not been fully examined.Methods: We applied ischemia-reperfusion (IR) to induce renal vascular injury and renal fibrosis in mice. Platelet-derived growth factor receptor β (PDGFR-β)-DTR-positive mice were generated to deplete pericytes, and exogenous b-pEPCs and the PDGFR-β ligand, PDGF chain B (PDGF-BB), were employed to explore the relationship among b-pEPCs, pericytes, vascular repair, and early renal fibrosis.Results: Administration of b-pEPCs reduced IR-induced pericyte-endothelial detachment, pericyte proliferation, and myofibroblast transition via a paracrine mode, which preserved not only vascular stabilization but also ameliorated IR-initiated renal fibrosis. PDGF-BB upregulated the expression of PDGFR-β, exacerbated vascular abnormality, and pericyte-myofibroblast transition, which were ameliorated by b-pEPCs administration. The exogenous b-pEPCs and their culture medium (CM) induced vascular injury protection, and renal fibrosis was blocked by selective deletion of pericytes.Conclusion: Exogenous b-pEPCs directly protect against IR-induced vascular injury and prevent renal fibrosis by inhibiting the activation of PDGFR-β-positive pericytes.

C16 Peptide Promotes Vascular Growth and Reduces Inflammation in a Neuromyelitis Optica Model.

The goal of this study was to elucidate the mechanism of action of C16, a laminin-1 peptide that competes with αvβ3 for integrin binding, in treating neuromyelitis optica (NMO). A NMO rat model was established and specific inhibitors were used to investigate the effect of Tie2 kinase, integrin, and PI3K/Akt signaling pathways on C16 function in NMO using histological, immunohistochemical, immunofluorescence, Western blot, and ELISA assays. A total of 150 rats were divided into five groups: a control untreated group (n = 18) and four test groups (n = 33 per group) including vehicle-treated control, C16, Tie2 kinase inhibitor + C16, and PI3K/Akt inhibitor LY294002 + C16. We found that inhibiting Tie2 kinase resulted in partial loss of C16 peptide-mediated effects, while suppressing PI3K/Akt signaling reduced C16 peptide-mediated effects. In addition, activation of the αvβ3 integrin axis and Tie2 kinase promoted PI3K/Akt signaling. Our study showed that the Tie2-PI3K/Akt, Tie2 integrin, and integrin-PI3K/Akt signaling pathways regulate C16 peptide function in vascular growth and stabilization as well as inflammation in NMO.

Angiogenic signaling in the lungs of a metabolically suppressed hibernating mammal (Ictidomys tridecemlineatus).

To conserve energy in times of limited resource availability, particularly during cold winters, hibernators suppress even the most basic of physiologic processes. Breathing rates decrease from 40 breaths/minute to less than 1 breath/min as they decrease body temperature from 37 °C to ambient. Nevertheless, after months of hibernation, these incredible mammals emerge from torpor unscathed. This study was conducted to better understand the protective and possibly anti-inflammatory adaptations that hibernator lungs may use to prevent damage associated with entering and emerging from natural torpor. We postulated that the differential protein expression of soluble protein receptors (decoy receptors that sequester soluble ligands to inhibit signal transduction) would help identify inhibited inflammatory signaling pathways in metabolically suppressed lungs. Instead, the only two soluble receptors that responded to torpor were sVEGFR1 and sVEGFR2, two receptors whose full-length forms are bound by VEGF-A to regulate endothelial cell function and angiogenesis. Decreased sVEGFR1/2 correlated with increased total VEGFR2 protein levels. Maintained or increased levels of key γ-secretase subunits suggested that decreased sVEGFR1/2 protein levels were not due to decreased levels of intramembrane cleavage complex subunits. VEGF-A protein levels did not change, suggesting that hibernators may regulate VEGFR1/2 signaling at the level of the receptor instead of increasing relative ligand abundance. A panel of angiogenic factors used to identify biomarkers of angiogenesis showed a decrease in FGF-1 and an increase in BMP-9. Torpid lungs may use VEGF and BMP-9 signaling to balance angiogenesis and vascular stability, possibly through the activation of SMAD signaling for adaptive tissue remodeling.

Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells.

Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.

Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability, we investigated the role of sEH in a mouse model of ROP. In WT mice, hyperoxia elicited tyrosine nitration and inhibition of sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP). Correspondingly, in a murine model of ROP, sEH-/- mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than did their WT littermates. Astrocytes were the cells most affected by sEH deletion, and hyperoxia increased astrocyte apoptosis. In rescue experiments, 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 and presenilin-1-associated protein to attenuate poly ADP-ribose polymerase activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss, but also reduced pathological vascular tuft formation in sEH-/- mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement for preventing retinopathy in preterm infants.

Systematic Identification of Cell-Cell Communication Networks in the Developing Brain.

SummarySince the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable “Brain interactome explorer”, available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

Angiopoietin-Tie Signaling Pathway in Endothelial Cells: A Computational Model.

SummaryThe angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2.

Inhibition of Jagged-Specific Notch Activation Reduces Luteal Angiogenesis and Causes Luteal Hemorrhaging of Hormonally Stimulated Ovaries.

Robust angiogenesis in the corpus luteum is critical for maintenance of pregnancy and thus mammalian female fertility. During angiogenesis, blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Pericytes are associated with capillaries and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial induction of Notch signaling in adjacent pericytes helps recruit and maintain pericyte coverage in some but not all tissue types. We have employed a Notch decoy, N110-24, which blocks Notch signaling in a ligand-specific manner, and determined that pharmacological inhibition of Notch ligand Jagged blocks luteal angiogenesis after normal ovulation, resulting in reduced luteal vasculature. Conversely, after ovarian hyperstimulation, a condition which occurs during fertility treatments, Jagged inhibition causes vascular dilation and hemorrhage. These results indicate that Jagged inhibition has effects in different ovarian angiogenic conditions, promoting vascular growth in the corpus luteum and vascular stability in hyperstimulated ovaries.

Lack of netrin-4 alters vascular remodeling in the retina.

Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina. We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG). Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio. These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.

Multimerin-2 maintains vascular stability and permeability

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2−/− (Mmrn2−/−) mice. Although Mmrn2−/− mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2−/− mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2−/− vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2−/− mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Thymosin Β4 Mediates Vascular Protection Via Regulation Of Low Density Lipoprotein Receptor Related Protein 1 (Lrp1)

Abstract Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic switch which promotes atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 (Tβ4) may additionally function to maintain healthy vasculature and protect against disease throughout postnatal life. This was supported by identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Tβ4-null mice display aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. We confirmed predisposition to disease in models of atherosclerosis and AAA. Diseased vessels and plaques were characterised by accelerated contractile-synthetic VSMC switching and augmented PDGFRβ signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with dysregulated endocytosis, leading to increased recycling of LRP1-PDGFRβ and reduced lysosomal targeting. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight which may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression.

P120 inhibits LPS/TNFα-induced endothelial Ang2 synthesis and release in an NF-κB independent fashion

Adherens junction protein p120 is thought to be crucial for maintaining vascular integrity, which is important in many pathologies and diseases including atherosclerosis, vascular malformations, hemorrhagic stroke, sepsis and others. However, the mechanisms responsible for this is not completely understood. In this study, using an unbiased proteomics approach, followed by other experimental techniques, we identified that in HUVECs p120 overexpression inhibits LPS/TNFα-induced angiopoietin-2 (Ang2) expression, a key switch of endothelial destabilization. Interestingly, p120 overexpression did not inhibit LPS/TNFα-induced expression of adhesion molecules/cytokines including VCAM-1, ICAM-1, E-selectin, MCP-1, IL-8 and IL-6 in our experimental system. Furthermore, this p120-mediated repression of Ang2 is in an NF-κB independent manner, possibly via transcription factor Ets1. Our results demonstrate that p120 influences vascular integrity by secreted signals, providing new insights into the mechanisms of p120-mediated vascular stability.