Obesity and leptin in breast cancer angiogenesis.

At the time of breast cancer diagnosis, most patients meet the diagnostic criteria to be classified as obese or overweight. This can significantly impact patient outcome: breast cancer patients with obesity (body mass index > 30) have a poorer prognosis compared to patients with a lean BMI. Obesity is associated with hyperleptinemia, and leptin is a well-established driver of metastasis in breast cancer. However, the effect of hyperleptinemia on angiogenesis in breast cancer is less well-known. Angiogenesis is an important process in breast cancer because it is essential for tumor growth beyond 1mm3 in size as well as cancer cell circulation and metastasis. This review investigates the role of leptin in regulating angiogenesis, specifically within the context of breast cancer and the associated tumor microenvironment in obese patients.

SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia. Methods: The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed. Results: The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy. Discussion: Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.

A Microphysiological HHT-on-a-Chip Platform Recapitulates Patient Vascular Lesions.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations focally develop in multiple organs. These can be small (telangiectasias) or large (arteriovenous malformations, AVMs) and may rupture leading to frequent, uncontrolled bleeding. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations for Endoglin (ENG) or Alk1 (ACVRL1), however, why loss of these genes manifests as vascular malformations remains poorly understood. To complement ongoing work in animal models, we have developed a microphysiological system model of HHT. Based on our existing vessel-on-a-chip (VMO) platform, our fully human cell-based HHT-VMO recapitulates HHT patient vascular lesions. Using inducible ACVRL1 (Alk1)-knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC) in the HHT-VMO. HHT-VMO vascular lesions develop over several days, and are dependent upon timing of Alk1 knockdown. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB expression implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring the positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that is sensitive to a treatment effective in patients. The VMO-HHT can be used to better understand HHT disease biology and identify potential new HHT drugs. Significance: This manuscript describes development of an organ-on-a-chip model of Hereditary Hemorrhagic Telangiectasia (HHT), a rare genetic disease involving development of vascular malformations. Our VMO-HHT model produces vascular malformations similar to those seen in human HHT patients, including small (telangiectasias) and large (arteriovenous malformations) lesions. We show that VMO-HHT lesions are sensitive to a drug, pazopanib, that appears to be effective in HHT human patients. We further use the VMO-HHT platform to demonstrate that there is a critical window during vessel formation in which the HHT gene, Alk1, is required to prevent vascular malformation. Lastly, we show that lesions in the VMO-HHT model are comprised of both Alk1-deficient and Alk1-intact endothelial cells.

Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.

A Predictive Model of Adaptive Resistance to BRAF/MEK Inhibitors in Melanoma.

The adaptive acquisition of resistance to BRAF and MEK inhibitor-based therapy is a common feature of melanoma cells and contributes to poor patient treatment outcomes. Leveraging insights from a proteomic study and publicly available transcriptomic data, we evaluated the predictive capacity of a gene panel corresponding to proteins differentially abundant between treatment-sensitive and treatment-resistant cell lines, deciphering predictors of treatment resistance and potential resistance mechanisms to BRAF/MEK inhibitor therapy in patient biopsy samples. From our analysis, a 13-gene signature panel, in both test and validation datasets, could identify treatment-resistant or progressed melanoma cases with an accuracy and sensitivity of over 70%. The dysregulation of HMOX1, ICAM, MMP2, and SPARC defined a BRAF/MEK treatment-resistant landscape, with resistant cases showing a >2-fold risk of expression of these genes. Furthermore, we utilized a combination of functional enrichment- and gene expression-derived scores to model and identify pathways, such as HMOX1-mediated mitochondrial stress response, as potential key drivers of the emergence of a BRAF/MEK inhibitor-resistant state in melanoma cells. Overall, our results highlight the utility of these genes in predicting treatment outcomes and the underlying mechanisms that can be targeted to reduce the development of resistance to BRAF/MEK targeted therapy.

Connexin 43-mediated neurovascular interactions regulate neurogenesis in the adult brain subventricular zone.

The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders.

Connexin37 Regulates Cell Cycle in the Vasculature.

Control of vascular cell growth responses is critical for development and maintenance of a healthy vasculature. Connexins - the proteins comprising gap junction channels - are key regulators of cell growth in diseases such as cancer, but their involvement in controlling cell growth in the vasculature is less well appreciated. Connexin37 (Cx37) is one of four connexin isotypes expressed in the vessel wall. Its primary role in blood vessels relies on its unique ability to transduce flow-sensitive signals into changes in cell cycle status of endothelial (and perhaps, mural) cells. Here, we review available evidence for Cx37's role in the regulation of vascular growth, vessel organization, and vascular tone in healthy and diseased vasculature. We propose a novel mechanism whereby Cx37 accomplishes this with a phosphorylation-dependent transition between closed (growth-suppressive) and multiple open (growth-permissive) channel conformations that result from interactions of the C-terminus with cell-cycle regulators to limit or support cell cycle progression. Lastly, we discuss Cx37 and its downstream signaling as a novel potential target in the treatment of cardiovascular disease, and we address outstanding research questions that still challenge the development of such therapies.

Regulation of Partial and Reversible Endothelial-to-Mesenchymal Transition in Angiogenesis.

During development and in several diseases, endothelial cells (EC) can undergo complete endothelial-to-mesenchymal transition (EndoMT or EndMT) to generate endothelial-derived mesenchymal cells. Emerging evidence suggests that ECs can also undergo a partial EndoMT to generate cells with intermediate endothelial- and mesenchymal-character. This partial EndoMT event is transient, reversible, and supports both developmental and pathological angiogenesis. Here, we discuss possible regulatory mechanisms that may control the EndoMT program to dictate whether cells undergo complete or partial mesenchymal transition, and we further consider how these pathways might be targeted therapeutically in cancer.

Slug regulates the Dll4-Notch-VEGFR2 axis to control endothelial cell activation and angiogenesis.

Slug (SNAI2), a member of the well-conserved Snail family of transcription factors, has multiple developmental roles, including in epithelial-to-mesenchymal transition (EMT). Here, we show that Slug is critical for the pathological angiogenesis needed to sustain tumor growth, and transiently necessary for normal developmental angiogenesis. We find that Slug upregulation in angiogenic endothelial cells (EC) regulates an EMT-like suite of target genes, and suppresses Dll4-Notch signaling thereby promoting VEGFR2 expression. Both EC-specific Slug re-expression and reduced Notch signaling, either by γ-secretase inhibition or loss of Dll4, rescue retinal angiogenesis in SlugKO mice. Conversely, inhibition of VEGF signaling prevents excessive angiogenic sprouting of Slug overexpressing EC. Finally, endothelial Slug (but not Snail) is activated by the pro-angiogenic factor SDF1α via its canonical receptor CXCR4 and the MAP kinase ERK5. Altogether, our data support a critical role for Slug in determining the angiogenic response during development and disease.

Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification.

Establishment of a functional vascular network is rate-limiting in embryonic development, tissue repair and engineering. During blood vessel formation, newly generated endothelial cells rapidly expand into primitive plexi that undergo vascular remodeling into circulatory networks, requiring coordinated growth inhibition and arterial-venous specification. Whether the mechanisms controlling endothelial cell cycle arrest and acquisition of specialized phenotypes are interdependent is unknown. Here we demonstrate that fluid shear stress, at arterial flow magnitudes, maximally activates NOTCH signaling, which upregulates GJA4 (commonly, Cx37) and downstream cell cycle inhibitor CDKN1B (p27). Blockade of any of these steps causes hyperproliferation and loss of arterial specification. Re-expression of GJA4 or CDKN1B, or chemical cell cycle inhibition, restores endothelial growth control and arterial gene expression. Thus, we elucidate a mechanochemical pathway in which arterial shear activates a NOTCH-GJA4-CDKN1B axis that promotes endothelial cell cycle arrest to enable arterial gene expression. These insights will guide vascular regeneration and engineering.

SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

FGF-dependent metabolic control of vascular development.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development.

The role of fluid shear stress in vasculature development and remodeling is well appreciated. However, the mechanisms regulating these effects remain elusive. We show that abnormal flow sensing in lymphatic endothelial cells (LECs) caused by Sdc4 or Pecam1 deletion in mice results in impaired lymphatic vessel remodeling, including abnormal valve morphogenesis. Ablation of either gene leads to the formation of irregular, enlarged and excessively branched lymphatic vessels. In both cases, lymphatic valve-forming endothelial cells are randomly oriented, resulting in the formation of abnormal valves. These abnormalities are much more pronounced in Sdc4-/-; Pecam1-/- double-knockout mice, which develop severe edema. In vitro, SDC4 knockdown human LECs fail to align under flow and exhibit high expression of the planar cell polarity protein VANGL2. Reducing VANGL2 levels in SDC4 knockdown LECs restores their alignment under flow, while VANGL2 overexpression in wild-type LECs mimics the flow alignment abnormalities seen in SDC4 knockdown LECs. SDC4 thus controls flow-induced LEC polarization via regulation of VANGL2 expression.

PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia.

Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

Isolation of Murine Embryonic Hemogenic Endothelial Cells.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Endothelial responses to fluid shear stress are essential for vascular development and physiology, and determine the formation of atherosclerotic plaques at regions of disturbed flow. Previous work identified VE-cadherin as an essential component, along with PECAM-1 and VEGFR2, of a complex that mediates flow signaling. However, VE-cadherin's precise role is poorly understood. We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex. VEGFR2 and VEGFR3 signal redundantly downstream of VE-cadherin. Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo. In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

Compromised regulation of tissue perfusion and arteriogenesis limit, in an AT1R-independent fashion, recovery of ischemic tissue in Cx40(-/-) mice.

Recently, we reported that recovery of tissue perfusion in the ischemic hindlimb was reduced, inflammatory response increased, and survival of distal limb tissue compromised in connexin 40 (Cx40)-deficient (Cx40(-/-)) mice. Here we evaluate whether genotype-specific differences in tissue perfusion, native vascular density, arteriogenesis, blood pressure, and chronic ANG II type 1 receptor (AT1R) activation contribute to poor recovery of ischemic hindlimb tissue in Cx40(-/-) mice. Hindlimb ischemia was induced in wild-type (WT), Cx40(-/-), and losartan-treated Cx40(-/-) mice by using surgical procedures that either maintained (mild surgery) or compromised (severe surgery) perfusion of major collateral vessels supplying the distal limb. Pre- and postsurgical hindlimb perfusion was evaluated, and tissue survival, microvascular density, and macrophage infiltration were documented during recovery. Hindlimb perfusion was compromised in presurgical Cx40(-/-) versus WT mice despite comparable native microvascular density. Hindlimb perfusion 24 h postsurgery in Cx40(-/-) and WT mice was comparable after mild surgery (collateral vessels maintained), but compromised arteriogenesis in Cx40(-/-) animals nevertheless limited subsequent recovery of tissue perfusion and compromised tissue survival. Prolonged pre- and postsurgical treatment of Cx40(-/-) mice with losartan (an AT1R antagonist) normalized blood pressure but did not improve tissue perfusion or survival, despite reduced macrophage infiltration. Thus it appears Cx40 is necessary for normal tissue perfusion and for recovery of perfusion, arteriogenesis, and tissue survival in the ischemic hindlimb. Our data suggest that Cx40(-/-) mice are at significantly greater risk for poor recovery from ischemic insult due to compromised regulation of tissue perfusion, vascular remodeling, and prolonged inflammatory response.

Connexin45 regulates endothelial-induced mesenchymal cell differentiation toward a mural cell phenotype.

OBJECTIVE: The focus of this study was to investigate the role of connexin (Cx) 45 in endothelial-induced mural cell differentiation. METHODS AND RESULTS: We created mural cell precursors that stably express only Cx45 in Cx43-deficient mesenchymal cells (ReCx45), and used our in vitro model of blood vessel assembly to assess the capacity of this Cx to support endothelial-induced mural cell differentiation. Lucifer Yellow dye injection and dual whole-cell patch clamping revealed that functional gap junctions exhibiting properties of Cx45-containing channels formed among ReCx45 transfectants, and between ReCx45 and endothelial cells. Heterocellular Cx45-containing gap junction channels enabled transforming growth factor-ß activation and promoted the upregulation of mural cell-specific proteins in the mesenchymal precursors. CONCLUSIONS: These studies reveal a critical role for Cx45 in the regulation of endothelial-induced mural cell differentiation, which is consistent with the phenotype of Cx45-deficient embryos that exhibit dysregulated transforming growth factor-ß and lack mural cell development.