Vascular Stability Research Articles

Retinoic Acid Regulates Endothelial β-catenin Expression and Pericyte Numbers in the Developing Brain Vasculature.

The acquisition of brain vascular properties, like tight junctions and pericytes, to form the blood-brain barrier (BBB) is crucial for a properly functioning central nervous system (CNS). Endothelial WNT signaling is a known driver of brain vascular development and BBB properties, however, it is unclear how endothelial WNT signaling is regulated. We recently showed that mouse embryos with disruptions in endothelial retinoic acid (RA) signaling have ectopic WNT signaling in the brain vasculature. Using immunohistochemistical analysis, we show that increased vascular WNT signaling in RA mutants (Pdgfbicre; dnRAR403-flox and Rdh10 mutants) is associated with elevated expression of the WNT transcriptional effector, β-catenin, in the brain endothelium. In vitro immunocytochemistry and proximity ligation studies in brain endothelial cells reveal that RA, through its receptor RARα, regulates β-catenin expression in brain endothelial cells via transcriptional suppression and phosphorylation events that targets β-catenin for proteasomal degradation, the latter dependent on PKCα. We find that one function of RA in regulating vascular WNT signaling is to modulate the pericyte numbers in the developing brain vasculature. RA-mediated regulation of vascular WNT signaling could be needed to prevent over-recruitment of pericytes that might impair endothelial-pericyte interactions crucial for vascular stability.

Collagen COL22A1 maintains vascular stability and mutations in COL22A1 are potentially associated with intracranial aneurysms.

ABSTRACTCollagen XXII (COL22A1) is a quantitatively minor collagen, which belongs to the family of fibril-associated collagens with interrupted triple helices. Its biological function has been poorly understood. Here, we used a genome-editing approach to generate a loss-of-function mutant in zebrafish col22a1. Homozygous mutant adults exhibit increased incidence of intracranial hemorrhages, which become more prominent with age and after cardiovascular stress. Homozygous col22a1 mutant embryos show higher sensitivity to cardiovascular stress and increased vascular permeability, resulting in a greater percentage of embryos with intracranial hemorrhages. Mutant embryos also exhibit dilations and irregular structure of cranial vessels. To test whether COL22A1 is associated with vascular disease in humans, we analyzed data from a previous study that performed whole-exome sequencing of 45 individuals from seven families with intracranial aneurysms. The rs142175725 single-nucleotide polymorphism was identified, which segregated with the phenotype in all four affected individuals in one of the families, and affects a highly conserved E736 residue in COL22A1 protein, resulting in E736D substitution. Overexpression of human wild-type COL22A1, but not the E736D variant, partially rescued the col22a1 loss-of-function mutant phenotype in zebrafish embryos. Our data further suggest that the E736D mutation interferes with COL22A1 protein secretion, potentially leading to endoplasmic reticulum stress. Altogether, these results argue that COL22A1 is required to maintain vascular integrity. These data further suggest that mutations in COL22A1 could be one of the risk factors for intracranial aneurysms in humans.

Low level laser therapy (LLLT) modulates ovarian function in mature female mice

It is known that LLLT has beneficial effects on several pathological conditions including wound healing, pain and inflammation. LLLT modulates biological processes, including cell proliferation, apoptosis and angiogenesis. In the present study, we examined the effect of local application of LLLT on follicular dynamics, ovarian reserve, AMH expression, progesterone levels, apoptosis, angiogenesis, and reproductive outcome in adult mice. LLLT (200 J/cm2) increased the percentage of primary and preantral follicles, whilst decreasing the percentage of corpora lutea compared to control ovaries. LLLT-treated ovaries did not exhibit any changes regarding the number of primordial follicles. We observed a higher percentage of AMH-positive follicles (in early stages of development) in LLLT-treated ovaries compared to control ovaries. LLLT reduced the P4 concentration and the apoptosis in early antral follicles compared to control ones. LLLT caused a reduction in the endothelial cell area and an increase in the periendothelial cell area in the ovary. Additionally, LLLT was able to improve oocyte quality. Our findings suggest that local application of LLLT modulates follicular dynamics by regulating apoptosis and the vascular stability in mouse ovary. In conclusion, these data indicate that LLLT might become a novel and useful tool in the treatment of several pathologies, including female reproductive disorders.

Role of ADTRP (Androgen-Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function.

BackgroundThe physiological function of ADTRP (androgen‐dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function.Methods and ResultsGenetic inhibition of Adtrp produced vascular malformations in the low‐pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE‐cadherin and claudin‐5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell‐based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low‐density lipoprotein receptor‐related protein 6 (LRP6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/β‐catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase (MMP)‐9 in endothelial cells and mast cells (MCs). Vascular lesions in newborn Adtrp −/− pups displayed accumulation of MCs, decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt‐pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling.ConclusionsOur studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt‐regulated genes that control vascular stability and integrity.

TNF-α-Induced YAP/TAZ Activity Mediates Leukocyte-Endothelial Adhesion by Regulating VCAM1 Expression in Endothelial Cells.

YAP/TAZ, a transcriptional co-activator of Hippo pathway, has emerged as a central player in vessel homeostasis such as sprouting angiogenesis and vascular barrier stabilization, during development. However, the role of YAP/TAZ in pathological angiogenesis remains unclear. Here, we demonstrated that YAP/TAZ is a critical mediator in leukocyte-endothelial adhesion induced by the vascular inflammatory cytokine TNF-α. YAP/TAZ was dephosphorylated, translocated from the cytosol to the nucleus, and activated by TNF-α in endothelial cells. A specific inhibitor of Rho GTPases suppressed the TNF-α-induced dephosphorylation of YAP. Knockdown of YAP/TAZ using siRNA significantly reduced the expression of the leukocyte adhesion molecule VCAM1 induced by TNF-α. The adhesion of monocytes to endothelial cells was also markedly reduced by YAP/TAZ silencing. However, knockdown of YAP/TAZ did not affect TNF-α-induced NF-κB signaling. Overall, these results suggest that YAP/TAZ plays critical roles in regulating TNF-α-induced endothelial cell adhesive properties without affecting the NF-κB pathway, and implicate YAP/TAZ as a potential therapeutic target for treating inflammatory vascular diseases.

Evaluation of aflibercept and ziv-aflibercept binding affinity to vascular endothelial growth factor, stability and sterility after compounding

PurposeTo investigate the binding affinity, stability, and sterility of aflibercept and ziv-aflibercept to vascular endothelial growth factor (Holash et al. in Proc Natl Acad Sci USA 99(17):11393–11398, 2002. https://doi.org/10.1073/pnas.172398299) after compounding and storage for up to 28 days at 4 °C and − 8 °C.MethodsTuberculin-type 1-mL syringes were prepared containing aflibercept (40 mg/mL) and ziv-aflibercept (25 mg/mL). Samples were stored at 4 °C and − 8 °C for 0, 14, and 28 days and evaluated for the binding affinity of anti-VEGF to VEGF and stability using enzyme-linked immunosorbent assays. The evaluation of sample sterility was performed.ResultsLaboratory trials with aflibercept and ziv-aflibercept showed preservation of the drug-binding capability to recombinant VEGF when stored in plastic syringes for up to 28 days at 4 °C and − 8 °C. No significant decrease in mass or concentration were observed. Microbiologic evaluations did not detect contamination in the syringes.ConclusionsThe current study corroborates that compounded anti-VEGF drugs aflibercept and ziv-aflibercept do not loose stability or binding affinity and do not become contaminated if prepared under sterile conditions and stored at 4 °C or − 8 °C for 14 or 28 days.

Role of angiopoietins in mesothelioma progression

Background and objectiveAnti-angiogenic treatment has been recently shown to be clinically beneficial for mesothelioma patients. Angiopoietins-1 and -2 are key regulators of tumor angiogenesis. Ang-1 is mainly known to promote angiogenesis and vessel stability, while Ang-2 could serve as an antagonist of Ang-1 causing vessel regression and destabilization or enhance angiogenesis in a context-dependent manner. We hypothesized that Ang-1 would promote and Ang2 would halt experimental mesothelioma by affecting tumor angiogenesis. MethodsTo examine the effects of angiopoietins in mesothelioma angiogenesis and in vivo growth we constructed Ang-1 or Ang-2 overexpressing AE17 and AB1 mesothelioma cells and implanted them in the respective syngeneic animals. We also explored the clinical relevance of our observations using the human tumoral mRNAseq data available in the TCGA database. Results and conclusionsAng-1 promotes mesothelioma angiogenesis and growth while the effect of Ang-2 is context-dependent. Low Ang-1 levels in human mesotheliomas are associated with the epitheloid subtype. Tumors of high Ang-1, or concurrent high Ang-2 and VEGF expression present high PECAM-1 and CDH5 expression, markers of vascularity and vascular stability, respectively. Our results highlight the importance of angiopoietins in mesothelioma pathophysiology and pave the way for the clinical development of novel anti-angiogenic strategies.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

COMP-Angiopoietin-1 accelerates muscle regeneration through N-cadherin activation

Angiopoietin-1 modulates vascular stability via Tie2 on endothelial cells. In our previous study, we also showed it acts as an inhibitor of cardiomyocyte death. However, it remains poorly understood how Ang1 regulates myogenesis during muscle regeneration. Here we found that COMP-Ang1 (cAng1) enhances muscle regeneration through N-cadherin activation. Muscle fiber regeneration after limb muscle damage by ischemic injury was enhanced with cAng1 treatment. Mechanistically cAng1 directly bound to N-cadherin on the myoblast surface in a Ca2+ dependent manner. The interaction enhanced N-cadherin activation via N-cadherin/p120-catenin complex formation, which in turn activated p38MAPK (but not AKT or ERK) and myogenin expression (but not myoD) as well as increasing myogenin+ cells in/ex vivo. After transplantation of GFP-expressing myoblasts (GFP-MB), we showed an increased generation of GFP+ myotubes with adenovirus cAng1 (Adv-cAng1) injection. Adv-cAng1, however, could not stimulate myotube formation in N-cadherin-depleted GFP-MB. Taken together, this study uncovers the mechanism of how cAng1 promotes myoblast differentiation and muscle regeneration through the N-cadherin/p120-catenin/p38MAPK/myogenin axis.

Long noncoding RNA TCONS_00024652 regulates vascular endothelial cell proliferation and angiogenesis via microRNA-21.

Acute coronary syndrome caused by the rupture of atherosclerotic plaques is one of the primary causes of major cardiovascular events, and neovascularization within the plaque is closely associated with its stability. Long noncoding RNA (lncRNAs) is a type of noncoding RNA that serves a crucial role in regulating vascular endothelial cells (VECs). The aim of the present study was to investigate the effect of lncRNA TCONS_00024652 on the proliferation and angiogenesis of VECs following stimulation with TNF-α. The expression of lncRNA and miRNA was measured in human umbilical vein endothelial cells (HUVECs) by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was measured using a Cell Counting Kit-8 assay. Wound healing and tube formation assays were performed to determine cell migration and angiogenesis. Interactions between TCONS_00024652 and miR-21 were determined using bioinformatics and a dual-luciferase reporter assay. The results demonstrated that TCONS_00024652 is highly expressed in TNF-α-induced HUVECs. Functional assays demonstrated that the dysregulated expression of TCONS_00024652 promotes endothelial cell proliferation and angiogenesis, whereas TCONS_00024652 knockdown induces the opposite effects. Bioinformatics analysis using starBase predicted putative binding at the 3′-untranslated region of TCONS_00024652 and miR-21 and luciferase reporter assays further verified this interaction. The results of the present study suggest that the targeting of TCONS_00024652 by miR-21 may be a potential method of improving vascular endothelial dysfunction, neovascularization maturation and plaque stabilization.

CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions

Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4+ and Rab7+ vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1+), Rab4+, Rab11+ , and Rab7+ vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

Neutrophil elastase inhibition effectively rescued angiopoietin-1 decrease and inhibits glial scar after spinal cord injury

After spinal cord injury (SCI), neutrophil elastase (NE) released at injury site disrupts vascular endothelium integrity and stabilization. Angiopoietins (ANGPTs) are vascular growth factors that play an important role in vascular stabilization. We hypothesized that neutrophil elastase is one of the key determinants of vascular endothelium disruption/destabilization and affects angiopoietins expression after spinal cord injury. To test this, tubule formation and angiopoietins expression were assessed in endothelial cells exposed to different concentrations of recombinant neutropil elastase. Then, the expression of angiopoietin-1, angiopoietin-2, and neutrophil elastase was determined at 3 h and at 1, 3, 5, 7, 14, 21, and 28 days in a clinically relevant model of moderate compression (35 g for 5 min at T10) spinal cord injury. A dichotomy between the levels of angiopoietin-1 and angiopoietin-2 was observed; thus, we utilized a specific neutrophil elastase inhibitor (sivelestat sodium; 30 mg/kg, i.p., b.i.d.) after spinal cord injury. The expression levels of neutropil elastase and angiopoietin-2 increased, and that of angiopoietin-1 decreased after spinal cord injury in rats. The sivelestat regimen, optimized via a pharmacokinetics study, had potent effects on vascular stabilization by upregulating angiopoietin-1 via the AKT pathway and preventing tight junction protein degradation. Moreover, sivelestat attenuated the levels of inflammatory cytokines and chemokines after spinal cord injury and hence subsequently alleviated secondary damage observed as a reduction in glial scar formation and the promotion of blood vessel formation and stabilization. As a result, hindlimb locomotor function significantly recovered in the sivelestat-treated animals as determined by the Basso, Beattie, and Bresnahan scale and footprint analyses. Furthermore, sivelestat treatment attenuated neuropathic pain as assessed by responses to von Frey filaments after spinal cord injury. Thus, our result suggests that inhibiting neutropil elastase by administration of sivelestat is a promising therapeutic strategy to inhibit glial scar and promote functional recovery by upregulating angiopoietin-1 after spinal cord injury.

Roles of fibronectin isoforms in neonatal vascular development and matrix integrity.

Fibronectin (FN) exists in two forms—plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.

Chronic mild hypoxia increases expression of laminins 111 and 411 and the laminin receptor α6β1 integrin at the blood-brain barrier

The laminin family of glycoproteins are major constituents of the basal lamina of blood vessels, and play a fundamental role in promoting endothelial differentiation and blood-brain barrier (BBB) stability. Chronic mild hypoxia (CMH), in which mice are exposed to 8% O2 for two weeks, induces a strong vascular remodeling response in the central nervous system (CNS) that includes endothelial proliferation, angiogenesis, arteriogenesis as well as increased expression of tight junction proteins, suggestive of enhanced vascular integrity. As previous studies highlight an important role for laminin in promoting vascular differentiation and BBB stability, the goal of this study was to determine if CMH influences the expression of the laminins and their cell surface receptors in cerebral blood vessels. Our studies revealed that over a 14 day period of CMH, blood vessels in the brain showed strong upregulation of the specific laminin subunits α1 and α4, corresponding to increased expression of laminins 111 and 411 respectively, with no discernible changes in the expression levels of the α2 or α5 laminin subunits. This was accompanied by marked endothelial upregulation of the laminin receptor α6β1 integrin but no alterations in the other laminin receptors α1β1 integrin or dystroglycan. In light of the instructive role for laminins in promoting vascular differentiation and stability, these data suggest that upregulation of the laminin-α6β1 integrin axis is part of the molecular response triggered by mild hypoxia that leads to enhanced BBB stability.

Chemo-manipulation of tumor blood vessels by a metal-based anticancer complex enhances antitumor therapy

Human pleural mesothelioma is an incurable and chemoresistant cancer. Using a nude mouse xenograft model of human pleural mesothelioma, we show that RAPTA-T, a compound undergoing preclinical evaluation, enhances tumor vascular function by decreasing blood vessel tortuosity and dilation, while increasing the coverage of endothelial cells by pericytes and vessel perfusion within tumors. This in turn significantly reduces the interstitial fluid pressure and increases oxygenation in the tumor. Consequently, RAPTA-T pre-treatment followed by the application of cisplatin or liposomal cisplatin (Lipoplatin) leads to increased levels of the cytotoxin in the tumor and enhanced mesothelioma growth inhibition. We demonstrate that the vascular changes induced by RAPTA-T are related, in part, to the inhibition of poly-(ADP-ribose) polymerase 1 (PARP-1) which is associated to tumor vascular stabilization. These findings suggest novel therapeutic implications for RAPTA-T to create conditions for superior drug uptake and efficacy of approved cytotoxic anti-cancer drugs in malignant pleural mesothelioma and potentially other chemoresistant tumors.

Abstract 924: A mathematical-experimental approach for predicting host responses in a preclinical model for trastuzumab-treated HER2+ breast cancer

Abstract Introduction: Trastuzumab, a targeted therapy for human epidermal growth factor receptor 2 (HER2) positive breast cancer, induces cell cycle arrest and inhibits HER2 expression. Trastuzumab has been shown to improve vascular delivery of subsequent cytotoxic therapies, but the mechanism by which it regulates tumor-associated angiogenesis is not well characterized. Therefore, we developed an integrated, mathematical-experimental approach to systematically investigate the interactions of tumor-growth characteristics—vasculature, immune response, hypoxia, and necrosis—and to evaluate their effects on tumor response to treatment in a murine model of HER2+ breast cancer. Experimental: Mice (N=102) were injected subcutaneously with HER2-overexpressing BT474 breast cancer cells and treated with trastuzumab (10 mg/kg) or saline. Tumor volumes for two separate cohorts of treated and control mice were recorded longitudinally, and either mice were quantitatively imaged or tumors extracted for histology over seven days. Necrosis, vasculature, and hypoxia were characterized by: immunohistochemistry for percent necrosis, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for the percentage of well-vascularized tissue, and 18F-fluoromisonidazole positron emission tomography (FMISO-PET) for percent hypoxia. Preliminary immune (myeloid) cell infiltration was quantified using immunofluorescence of F4/80 and CD11c. Mathematical Model: A system of five coupled, ordinary differential equations describing the temporal variation in tumor growth, vasculature, hypoxia, necrosis, and immune response was calibrated using data for tumor volume and percentages of well-vascularized tissue, hypoxia, and necrosis. After validation using a second tumor volume data set, uncertainty analysis was used to verify plausible overlap between the model's simulations and the experimental data when considering calibration error, and sensitivity analysis identified critical parameters for experimental estimation and potential model reductions. Results and Discussion: The calibrated model was used to predict immune response behavior over time and yielded different results between the two groups—stagnant versus increasing immune component values for control versus treated mice, respectively. Preliminary immunofluorescence data support the immunological predictions of the model; in particular, F4/80 and CD11c co-staining in the total tissue is greater in trastuzumab treated than control tumors (p < 0.03). Furthermore, differences in the calibrated parameter sets indicate several experimentally testable hypotheses, including increased cross-talk between immune cells and vasculature as a mechanism for vascular stabilization due to trastuzumab therapy. We acknowledge the support of CPRIT RR160005 and NCI R01CA186193. Citation Format: Angela M. Jarrett, Meghan Bloom, Wesley Godfrey, Anum Syed, David A. Ekrut, Lauren I. Ehrlich, Thomas E. Yankeelov, Anna G. Sorace. A mathematical-experimental approach for predicting host responses in a preclinical model for trastuzumab-treated HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 924.

Abstract 5067: b1-Integrin promotes inflammatory cytokine-induced vascular leakage

Abstract Increased capillary leakage is a potentially life-threatening condition associated with sepsis and cytokine release syndrome (CRS) that develops as a side effect of novel chimeric antigen receptor (CAR) T-cell cancer immunotherapies. CRS and sepsis are characterized by immune activation, resulting in elevated circulating levels of inflammatory cytokines including IL-6 and IL-1β, and thrombin that promote vascular leakage and the development of hypovolemic shock and multiorgan failure. However, molecular mechanisms behind capillary leakage remain unknown, and importantly, targeted approaches for the management of capillary leakage are limited. Here, we found that β1-integrin acts as a critical universal mediator of endothelial permeability induced by various inflammatory agents, including LPS, IL-6, IL-1β and thrombin. In a mouse model of LPS-induced systemic inflammation, resulting in increased inflammatory cytokine production and vascular leakage, function blocking antibodies against β1-integrin prevented vascular leakage and improved integrity of vascular endothelium, leading to protection from LPS-induced cardiac failure. β1-integrin blocking antibodies were effective in reducing vascular leakage even when administered after the onset of systemic inflammation and increase in the serum levels of IL-6, IL-1β and TNF-α. By using an in vitro model, we found that inflammatory agents generated mechanical stress, in a β1-integrin dependent manner, leading to endothelial cell contractility and permeability. Conclusively, our data indicates that β1-integrin promotes vascular leakage in inflammation, and that targeting this mechanism may have therapeutic utility for vascular stabilization in CRS and capillary leak syndromes associated with immune activation and systemic inflammation. Citation Format: Elina A. Kiss, Laura Hakanpää, Guillaume Jacquemet, Ilkka Miinalainen, Martina Lerche, Camilo Guzman, Eero Mervaala, Lauri Eklund, Johanna Ivaska, Pipsa Saharinen. b1-Integrin promotes inflammatory cytokine-induced vascular leakage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5067.

Targeting β1-integrin inhibits vascular leakage in endotoxemia.

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak.

Platelet-Derived Growth Factor Subunit B Signaling Promotes Pericyte Migration in Response to Loud Sound in the Cochlear Stria Vascularis.

Normal blood supply to the cochlea is critical for hearing. Noise damages auditory sensory cells and has a marked effect on the microvasculature in the cochlear lateral wall. Pericytes in the stria vascularis (strial pericytes) are particularly vulnerable and sensitive to acoustic trauma. Exposure of NG2DsRedBAC transgenic mice (6-8weeks old) to wide-band noise at a level of 120dB for 3h per day for 2 consecutive days produced a significant hearing threshold shift and caused pericytes to protrude and migrate from their normal endothelial attachment sites. The pericyte migration was associated with increased expression of platelet-derived growth factor beta (PDGF-BB). Blockade of PDGF-BB signaling with either imatinib, a potent PDGF-BB receptor (PDGFR) inhibitor, or APB5, a specific PDGFRβ blocker, significantly attenuated the pericyte migration from strial vessel walls. The PDGF-BB-mediated strial pericyte migration was further confirmed in an in vitro cell migration assay, as well as in an in vivo live animal model used in conjunction with confocal fluorescence microscopy. Pericyte migration took one of two different forms, here denoted protrusion and detachment. The protrusion is characterized by pericytes with a prominent triangular shape, or pericytes extending fine strands to neighboring capillaries. The detachment is characterized by pericyte detachment and movement away from vessels. We also found the sites of pericyte migration highly associated with regions of vascular leakage. In particular, under transmission electron microscopy (TEM), multiple vesicles at the sites of endothelial cells with loosely attached pericytes were observed. These data show that cochlear pericytes are markedly affected by acoustic trauma, causing them to display abnormal morphology. The effect of loud sound on pericytes is mediated by upregulation of PDGF-BB. Normal functioning pericytes are required for vascular stability.

Angiogenesis and vascular stability in eicosanoids and cancer.

Angiogenesis and inflammation are hallmarks of cancer. Arachidonic acid and other polyunsaturated fatty acids (PUFAs) are primarily metabolized by three distinct enzymatic systems initiated by cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes (CYP) to generate bioactive eicosanoids, including prostanoids, leukotrienes, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids. As some of the PUFA metabolites playing essential roles in inflammatory processes, these pathways have been widely studied as therapeutic targets of inflammation. Because of their anti-inflammatory effects, these pathways were also proposed as anti-cancer targets. However, although the eicosanoids were linked to endothelial cell proliferation and angiogenesis almost two decades ago, it is only recently PUFA metabolites, especially those generated by CYP enzymes and the soluble epoxide hydrolase (sEH), have been recognized as important signaling mediators in physiological and pathological angiogenesis. Despite the fact that tumor growth and invasion are heavily dependent on inner-tumor angiogenesis and influenced by vascular stability, the role played by PUFA metabolites in tumor angiogenesis and vessel integrity has beenlargely overlooked. This review highlights current knowledge on the function of PUFA metabolites generated by the CYP/sEH pathway in angiogenesis and vascular stability as well astheir potential involvement in cancer development.