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. Author manuscript; available in PMC: 2025 Apr 15.
Published in final edited form as: Compr Physiol. 2024 Mar 29;14(2):5389–5406. doi: 10.1002/cphy.c230014

A TRP to Pathological Angiogenesis and Vascular Normalization

Venkatesh Katari 1, Kesha Dalal 1, Ravi K Adapala 1, Brianna D Guarino 2,3, Narendrababu Kondapalli 1, Sailaja Paruchuri 1, Charles K Thodeti 1,*
PMCID: PMC11998386  NIHMSID: NIHMS2071987  PMID: 39109978

Abstract

Uncontrolled angiogenesis underlies various pathological conditions such as cancer, age-related macular degeneration (AMD), and proliferative diabetic retinopathy (PDR). Hence, targeting pathological angiogenesis has become a promising strategy for the treatment of cancer and neovascular ocular diseases. However, current pharmacological treatments that target VEGF signaling have met with limited success either due to acquiring resistance against anti-VEGF therapies with serious side effects including nephrotoxicity and cardiovascular-related adverse effects in cancer patients or retinal vasculitis and intraocular inflammation after intravitreal injection in patients with AMD or PDR. Therefore, there is an urgent need to develop novel strategies which can control multiple aspects of the pathological microenvironment and regulate the process of abnormal angiogenesis. To this end, vascular normalization has been proposed as an alternative for antiangiogenesis approach; however, these strategies still focus on targeting VEGF or FGF or PDGF which has shown adverse effects. In addition to these growth factors, calcium has been recently implicated as an important modulator of tumor angiogenesis. This article provides an overview on the role of major calcium channels in endothelium, TRP channels, with a special focus on TRPV4 and its downstream signaling pathways in the regulation of pathological angiogenesis and vascular normalization. We also highlight recent findings on the modulation of TRPV4 activity and endothelial phenotypic transformation by tumor microenvironment through Rho/YAP/VEGFR2 mechanotranscriptional pathways. Finally, we provide perspective on endothelial TRPV4 as a novel VEGF alternative therapeutic target for vascular normalization and improved therapy.

GRAPHICAL ABSTRACT

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Introduction

Angiogenesis is a coordinated process orchestrated by endothelial cells (EC) to form vascular networks in high-demanding growing tissues (63). This tight regulation of angiogenesis is controlled by several soluble and mechanical factors that lie within the microenvironment of vascular tissue. These soluble factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), angiopoietin, etc., and mechanical forces such as shear stress (tangential frictional force due to blood flow), stress force (exerted by the skeletal muscle activity), and tensile stress (circumferential force acting on vessel wall due to stretch of the vessel wall) (63). The most well-characterized soluble secreted factor implicated in angiogenesis is VEGF, which acts through three different VEGF receptors, VEGFR1, 2, and 3. Adult human tissues express low levels of VEGF whereas, naive fetal and placental tissues express extremely high amounts of VEGF due to high oxygen demand and the formation of new blood vessels (18). VEGF production is highly dependent on surrounding oxygen concentration. Low oxygen tension (hypoxia) stimulates the production of VEGF through the factor known as hypoxia-inducible factor 1-alpha (HIF1α) (51). HIF1α induces the expression of pro-angiogenic factor VEGF-A via binding to VEGF-A promoter (6, 67).

Particularly, VEGF-A165 binds to its receptor VEGFR2 and promotes VEGFR2 dimerization, autophosphorylation of tyrosine residues at the carboxy-terminal domain (30, 121), and triggers various biological responses such as vascular permeability, migration, proliferation, and survival of EC. There are five major phosphorylation sites in VEGFR2: Y951, Y1054, Y1059, Y1175, and Y1214 and each one critically dictates specific cellular functions including cell proliferation, vascular permeability, migration, actin remodeling, and focal adhesions (1, 121). These sites also create binding sites for various other kinases such as PI3K, PLCγ, and Src kinase (77, 106). VEGFR2 translocates from Golgi to plasma membrane and upon binding with VEGF, triggers a plethora of downstream pathways including Raf-MEK-ERK (cell proliferation), PKC/eNOS/NO (cell permeability), Src/PI3K/AKT/BAD/Caspase9 (cell survival), and NCK/p38/MAPKAPK2/3 (cell migration) which then lead to tube formation and angiogenesis (92, 105) (Figure 1). PDGF is another angiogenic factor that shares 21% to 24% amino acid sequence homology and 8 conserved cysteine domains are found identical with the VEGF amino acid sequence (58, 116). Moreover, PDGFB/PDGFR-β signaling has been demonstrated in the maturation of the vascular wall; however, the molecular mechanisms that regulate physiological angiogenesis are not fully explored (98). Finally, these growth factors coupled with extracellular matrix (ECM), foster EC proliferation, and migration, and form tube-like structures. Mural cells (pericytes and smooth muscle cells) are recruited to the abluminal surface of the endothelium that then establish blood flow and regulate the physiological angiogenesis process (91).

Figure 1.

Figure 1

Activation of VEGFR2 and its downstream signaling mechanisms for angiogenesis. Binding of VEGF to VEGFR2, a protein tyrosine kinase receptor results in autophosphorylation (activation) at various tyrosine residues such as Y951, Y1054, Y1059, Y1175, and Y1214. VEGFR2 autophosphorylation, in turn, activates multiple signal transduction molecules such as phosphoinositide 3-kinase (PI3K), phospholipase C gamma (PLCγ), Src kinase (Src), and phosphatidylinositol biphosphate (PIP2), which mediates downstream effects of VEGFR2 including cell survival, actin reorganization, migration, and proliferation that are required for angiogenesis. VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase; PLCγ, phospholipase C -γ; DAG, diacylglycerol; IP3, inositol (1,4,5)-triphosphate; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; FAK, focal adhesion kinase; PI3K, phosphoinositide 3-kinase; p38 MAPK, p38 mitogen-activated protein; Erk, extracellular regulated kinase; HSP27, heat shock protein; EC, endothelial cell. Figure created with Biorender.com.

In 1971, Folkman, for the first time, proposed that angiogenesis plays an important role in tumor growth and metastasis and blocking angiogenesis could be a viable strategy to halt tumor growth (33). The growth-promoting signals are at the highest level in tumor microenvironment and induce uncontrolled cell division, sprouting, and migration of EC. Cells in the tumor microenvironment crave for excessive oxygen and nutrients for continuous use in the remodeling of the tumor vasculature, which then becomes leaky and causes irregular blood flow (33, 127). Leaky basal lamina of the tumor vessels recruits activated platelets, which in turn release PDGF. PDGF activates perivascular cells and increases coverage and vascular stability without proper maturation due to persistent stimulatory signals, which further leads to abnormal vessel formation (47). Furthermore, the tumor tissues, which overexpress angiopoietin-2 (ANGPT2), demonstrate increased vascularization and perfusion, leading to destabilization of the ECM and endothelium (62). These pro-angiogenic factors such as VEGF, bFGF, Ang-2, and PDGF activate their respective tyrosine kinase receptors and regulate tumor angiogenesis. The binding of these growth factors to their receptors activates several signaling molecules including phosphatidylinositol 3-kinase (PI3Kinase), signal transducer and activator of transcription (STAT), and receptor tyrosine kinases (RTKs). Activation of these molecules triggers various cellular processes such as cell cycle, migration, and proliferation (41). The concept of angiogenesis inhibition led to the development of antiangiogenic therapy in patients with several types of cancers. The United States Food and Drug Administration (FDA) has approved several inhibitors of angiogenesis to treat various types of cancers. Most of these were developed specifically to target VEGF, and/or its receptors. Various FDA-approved angiogenesis inhibitors and their mode of action are listed in Table 1. Several side effects of VEGF-targeting angiogenesis inhibitors are observed in cancer patients including hemorrhage, clots in the arteries (with resultant stroke or heart attack), hypertension, impaired wound healing, reversible posterior leukoencephalopathy syndrome (a brain disorder), and protein in the urine (64, 101, 104). Antiangiogenic agents that target the VEGF receptor also have additional side effects, including fatigue, diarrhea, biochemical hypothyroidism, hand-foot syndrome, cardiac failure, and hair changes (16, 50, 111, 117, 119). Therefore, there is an urgent need to develop an alternative strategy to treat deadly cancerous diseases.

Table 1.

FDA (Food and Drug Administration) Approved Drugs Used in Clinical Trials as an Antiangiogenic Therapy

FDA-approved drugs Clinical condition Mode of action Clinical outcome Adverse events References
Bevacizumab Colorectal cancer Selectively binds to circulating VEGF and inhibits their interaction with VEGF receptor present on cell surfaces. This inhibition leads to a reduction in microvascular tumor growth Clinical improvement in survival among patients with metastatic colorectal cancer Hypertension, proteinuria, gastrointestinal perforation, and other side effects include thrombosis and bleeding Hurwitz et al. (50) and Nalluri, Chu, Keresztes, Zhu, and Wu (85)
Axitinib Renal cell carcinoma Potent inhibitor of vascular endothelial growth factor receptors (VEGFR)-1, 2, and 3 Tumor shrinkage was observed and improved clinical activity Hypertension, proteinuria, and hand-foot syndrome are more common in Japanese patients than in Western patients Tomita et al. (117)
Cabozantinib Hepatocellular carcinoma Inhibits tyrosine kinases, including vascular endothelial growth factor receptors 1, 2, and 3, MET, and AXL Significantly improvement in survival rate Palmar-plantar erythrodysesthesia, hypertension, increased aspartate aminotransferase level, fatigue, and diarrhea Abou-Alfa et al. (2)
Everolimus Pancreatic neuroendocrine tumor mTOR inhibitor 65% reduction in the estimated risk of progression or death Stomatitis, rash, diarrhea, fatigue, and upper respiratory infections are observed Lane et al. (64) and Yao et al. (137)
Lenalidomide Chronic myelomonocytic leukemia Inhibits VEGF-induced PI3K-Akt pathway signaling Partial remission in one patient and stable disease in nine patients Lack of response in three patients and thrombocytopenia Burgstaller et al. (16)
Lenvatinib mesylate Biliary tract cancer Kinase inhibitor for vascular endothelial growth factor receptors (VEGFR)-1, 2, and 3 Tolerable safety profile in patients Grade ≥3 treatment-emergent adverse events (TEAEs) occurred in 21 patients included hypertension, proteinuria Ueno et al. (119)
Pazopanib Metastatic clear cell renal cell carcinoma Inhibits epidermal growth factor receptor tyrosine kinases Decreases mortality Diarrhea, hypertension, and hair color change remained the most common adverse events Sternberg et al. (111)
Ramucirumab Adenocarcinoma Monoclonal antibody, vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor Improves survival as second-line treatment of patients when combined with paclitaxelor erlotinib or docetaxel Dermatitis acneiform, increased alanine aminotransferase, pneumonia, neutropenia, leucopenia, hypertension, fatigue, anemia, and abdominal pain Nakagawa et al. (84) and Wilke et al. (131)
Regorafenib Hepatocellular carcinoma Multi-kinase inhibitor. Inhibitor of angiogenic (VEGFR1–3, TIE2), stromal (PDGFR-β, FGFR), and oncogenic receptor tyrosine kinases (KIT, RET, and RAF) Improves overall survival in patients Hypertension, hand-foot skin reaction, and diarrhea Bruix et al. (15)
Sorafenib Hepatocellular carcinoma Multi-kinase inhibitors, including VEGFR, PDGFR, and RAF kinases Significantly improved progression-free survival when combined with transarterial chemoembolisation (TACE) Palmar-plantar erythrodysesthesia, decreased appetite, and anemia Kudo et al. (61)
Vandetanib Medullary thyroid cancer Inhibits tyrosine kinases including VEGFR2 and EGFR Improved therapeutic efficacy in a phase III trial of patients Common adverse events include diarrhea, rash, nausea, hypertension, and headache Wells et al. (129)

Tumor Microenvironment, Signaling Pathways, and Pathological Angiogenesis

The tumor microenvironment (TME) is a combination of both cellular and extracellular components that continuously drive tumorigenesis (11). The TME comprises a variety of constituents including immune cells, fibroblasts, blood vessels, ECM, and other signaling molecules (34, 110). Hypoxia and immune suppression are considered the hallmark functions of TME (35). Hypoxic environment significantly increases capillary permeability, release of fibrin from leaky vessels, and collagenase activation, thereby promoting ECM remodeling (95). In tumor tissues, tumor cells themselves form tubular structures for fluid transport, a process known as vasculogenic mimicry (29). These tubular structures attach to EC and increase neovascularization processes and promote tumor growth, invasion, and metastasis (54). TME modulates the immune system by inhibiting dendritic recruitment, maturation, and antigen presentation, thereby reducing the activity of cytotoxic T-cells through secreted angiogenic factors (54, 72). Various cells of the TME secrete angiogenic factors such as VEGF, FGF, PDGF, etc. These soluble factors aid in the formation of new blood vessels; however, these vessels fail to achieve final maturation and become hyperpermeable (41). The availability of soluble factors such as PDGF or VEGF in the TME dictates the functions of vascular smooth muscle cells (VSMC) and EC, respectively, and balances the physiological communication between these cells (78). VEGF initiates new blood vessel formation whereas PDGF induces VSMC coverage to newly formed blood vessels (128). Various signaling mechanisms play a prominent role in the development of pathological angiogenesis, which further activates their downstream intermediates such as RAF1-MEK-ERK1/2 (76), and PLCγ-PKC-PKD (133), which regulate cell proliferation and migration, respectively. The other downstream cascades PLCγ-eNOS-NFAT (136) and PLCγ-COX2-PGE2 (20) regulate cell survival, invasion, migration, tube formation, and angiogenesis.

Neovascularization and Ocular Diseases

Neovascularization is a critical factor for the pathogenesis of age-related wet macular degeneration (Wet AMD) and proliferative diabetic retinopathy (PDR) (74). Wet AMD is characterized by the rapid explosive growth of vessels from choroid into subretinal space (7, 43, 66). In contrast, hyperglycemia-induced damage of retinal endothelium in diabetes leads to ischemia/hypoxia which in turn increases angiogenic growth factors such as VEGF by hypoxia-inducible factor 1α (HIF1α) (27). As with tumor angiogenesis, VEGF/VEGFR2 signaling has been identified as the major player of neovascular ocular diseases and has become the target for therapy (74, 79). In fact, the anti-VEGF drugs Ranibizumab, Aflibercept, and Brolucizumab have been approved by FDA to treat Wet AMD and PDR and other tyrosine kinase inhibitor (TKI) drugs are in different stages of clinical trials (Table 1) (59). Despite the success of anti-VEGF therapy for these diseases, side effects such as retinal vasculitis and retinal inflammation have been reported (28) (Figure 2). In addition, a subset of patients exhibited unresponsiveness for the VEGF therapy (109) indicating a need for VEGF-alternative therapies.

Figure 2.

Figure 2

VEGF and TRPV4-based therapies for retinopathy. Retinopathy is characterized by explosive growth of blood vessels in the eye. Current treatments include intraocular injection of anti-VEGF drugs (for example; Lucentis). Although anti-VEGF injections reduce blood vessel growth; these patients still suffer from retinal vasculitis and retinal inflammation and in some cases are unresponsive to anti-VEGF therapy. We propose that application of TRPV4 activators as eyedrops could normalize retinal vasculature specifically when combined with other drugs that combat pathological angiogenesis. This application will also have additional beneficial effects such as reduction in intraocular pressure (IOP) and improved outflow facility. This novel therapeutic approach may altogether avoid repeated injections of anti-VEGF drugs into the eye. Figure created with Biorender.com.

Transient Receptor Potential Channels and Angiogenesis

On the other hand, one of the important second messengers released by VEGF/PDGF is calcium, which has been recently implicated in tumor angiogenesis (82). Although the exact channels that mediate calcium influx by VEGFR are not known, transient receptor potential (TRP) channels (Figure 3) were shown to activate VEGFR-2 and their downstream signaling molecules which regulate cell migration, proliferation, tube formation, and angiogenesis within the TME (5, 38, 56, 57, 87, 108, 114, 115).

Figure 3.

Figure 3

A schematic showing structural domains of TRPV4. TRPV4 is a tetramer, and each subunit consists of 6 transmembrane domains (S1-S6) with a pore loop between S5 and S6 domains. TRPV4 contains six ankyrin domains (ARD) and a proline-rich domain (PRD) in the N-terminus, which may serve as a path for mechanical force transfer via their indirect interactions with integrins and cytoskeleton. Figure created with Biorender.com.

The members of the TRP superfamily of channels are well studied for their several downstream mechanisms that dictate numerous cellular functions, including cell proliferation, migration, angiogenesis, and permeability (5, 108, 113115). The TRP superfamily is divided based on its sequence and topology into seven subfamilies including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystic), and TRPML (mucolipin). TRP ion channels are nonselective, which regulate the influx of calcium ions and thereby control angiogenesis (Figure 4A).

Figure 4.

Figure 4

TRPV4 mechanotransduction in endothelium and angiogenesis. (A) TRP channels and their role in angiogenesis. (B) TRPV4 is activated polymodally by endogenous (arachidonic acid-AA and its derivatives epoxyeicosatrienoic acids-EETs) or exogenous (GSK1016790A or 4-a-PDD) agonists or mechanical forces such as mechanical stretch, shear stress and matrix stiffness. Mechanical forces applied on ECM are transduced through β1 integrin/CD98 to TRPV4 resulting in the TRPV4-mediated calcium influx and activation of PI3K. PI3K in turn activates additional β1 integrins which regulate reorientation (directional migration) of the cytoskeleton and cell via Rho GTPases leading to permeability, vasodilation, and angiogenesis. Figure created with Biorender.com.

Transient Receptor Potential Canonical Channels

The isoforms of transient receptor potential canonical (TRPC) channels, such as TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 have shown involvement in cell proliferation, migration, tube formation, and angiogenesis (146). Na+-Ca2+ exchange (NCX) transporters play a critical role in regulating VEGF and its downstream signaling molecules. Pharmacological inhibition of NCX1 significantly reduces VEGF-induced ERK1/2 phosphorylation and inhibits EC proliferation, migration, and tube formation (8). TRPC3 functionally couples with NCX and activates ERK1/2 by phosphorylation, which then enhances the angiogenic process (9). TRPC5 knockdown or genetic deletion attenuates tube formation through retaining nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3) in the cytoplasm, and inhibiting angiopoietin-1 (ANGPT1) activity in the hypoxia-ischemia murine model (146). Similarly, TRPC6 has been demonstrated to regulate VEGF-mediated angiogenesis and pharmacological inhibition of TRPC6 in human umbilical vein endothelial cells (HUVECs) suppressed their proliferation and tube formation (36, 42). Altogether, TRPC channels positively regulate the angiogenic process by involving stromal interaction molecule-1 (STIM1) (10). Small interference RNA (siRNA) targeting TRPC channels significantly inhibited cell proliferation and differentiation in non-small cell lung cancer and ovarian cancer (53, 141). However, the role of TRPC channels in tumor angiogenesis and its progression is not fully explored (40, 123).

Transient Receptor Potential Melastatin Channels

Transient receptor potential melastatin (TRPM) channels are widely expressed in various malignant cancers such as prostate, pancreatic, breast, melanoma, and leukemia. These channels promote intracellular calcium signaling which triggers various downstream pathways and cellular functions (80). Inhibition of TRPM channels has been shown to improve capillary integrity and tube formation in various disease models and their activation has been shown to result in oncosis. Under hypoxic conditions, pharmacological or siRNA-based TRPM4 inhibition has been shown to increase tube formation both in vitro and in vivo (68). Blocking of TRPM4 protects neural tissue by promoting angiogenesis and has shown promise as a potential drug target for stroke therapy (68). Additionally, TRPM2 knockout mice have been shown to have aberrant angiogenesis and neovascularization in the hind limb ischemia model as compared to wild-type mice (80).

Transient Receptor Potential Ankyrin Channels

Transient receptor potential ankyrin (TRPA) channels also have angiogenic effects in the tumor environment. In prostate cancer, the antibacterial agent triclosan (TCS) activates TRPA1 and induces VEGF secretion. The secreted VEGF can potentiate various functions including vessel growth and sprouting from a preexisting lymphatic vessel in the lymphatic endothelium (lymphangiogenesis). Thus, TCS-induced TRPA1 activation may favor the advancement of prostate cancer by angiogenesis and metastasis (24).

Minimal studies have been published on TRPA, TRPN (NO-mechano-potential, NOMP), TRPP (polycystin), and TRPML (mucolipin) isoforms in tumor angiogenesis.

Transient Receptor Potential Vanilloid Channels

Transient receptor potential vanilloid (TRPV) channels have been identified as potent regulators of physiological and pathological angiogenesis (102). TRPV channels are associated with cancer progression by triggering cell proliferation, migration, angiogenesis, and invasion thus leading to uncontrolled expansion of tumor tissue (96, 97). Different isoforms of TRPV channels have been identified and are widely expressed, such as TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6 in various cell types, and show response to heat and taste of garlic (allicin), capsaicin, and piperine (19, 52). TRPV1 is a tetrameric protein specifically expressed in premalignant and low-grade papillary skin carcinoma, whereas its expression is substantially absent in invasive carcinoma. TRPV1 is known to exhibit tumor suppressive activity in mice skin carcinogenesis with significantly down-regulated expression of epidermal growth factor receptor (EGFR) (19). Anandamide (N-arachidonyl ethanolamide, AEA) induces EC proliferation and tube formation which is efficiently reduced by TRPV1 antagonist SB366791. Taken together AEA might promote tumor angiogenesis via TRPV1 (48). TRPV2 is over-expressed in urothelial cancer cells and controls its growth and progression by the IGF-1/IGF-1R pathway (145). Nabissi et al. demonstrated that TRPV2 channels negatively control glioma cell proliferation and resistance to fas-induced apoptosis in an ERK-dependent manner (83). Therefore, TRPV channels may regulate tumor angiogenesis and cancer progression at different levels (i) by acting through calcium influx mechanisms and regulating the expression and activity of cell-surface glycoproteins (ii) by regulating binding, trafficking, and functional activity of various proangiogenic factors (iii) by interacting with specific G protein-coupled receptors (GPCRs) at the plasma membrane and targeting different intracytoplasmic signaling pathways. As with many other TRPV channels, TRPV4 is a well-known regulator of tumor angiogenesis by involving cytoskeletal dynamics and Rho pathways (5, 115).

Transient Receptor Potential Vanilloid type 4 Channel

Transient receptor potential vanilloid type 4 (TRPV4) is a ubiquitously expressed channel and is shown to be mechanosensory in many cell types including EC (60, 113, 114). The location of the TRPV4 gene is on the long (q) arm of chromosome 12 at position 24.1. Botte et al. reported the structure of human TRPV4 using a Cryo-EM model in the presence of the archetypical phorbol acid agonist, 4α-PDD (14). These studies revealed that TRPV4 is a homotetramer and consists of six membrane-spanning domains (S1-S6) with a pore loop, an N-terminal domain with at least three ankyrin repeats (ARD), and a C-terminal domain residue within the cytoplasm (Figure 3). All these domains exhibit the basic function of the scaffold arrangement of the TRPV4 protein. TRPV4 ion channels are robustly activated by the specific TRPV4 agonist GSK1016790A (GSK101) and regulate calcium influx through the pore region.

TRPV4 Role in Normal Endothelium

TRPV4 in EC is activated by various chemical and physical stimuli, such as the GSK1016790A, 4αPDD, arachidonic acid, temperature (>27°C), hypotonic condition, pressure, shear stress, and membrane stretch; thus activated TRPV4, increases the substantial influx of calcium [Ca2+] into the endothelium (88) (Figure 4B). The activated TRPV4 ion channels are implicated in mechanotransduction and vascular-related functions such as vasodilation, vasoconstriction, vascular permeability, vascular remolding, vascular damage, and play important roles in vascular-related diseases (4, 138). Loot et al. demonstrated the role of epoxyeicosatrienoic acids (EETs) in flow-induced vasodilation. Cytochrome P450 (CYP) enzymes play a regulatory role in flow-mediated TRPV4 activation via EET generation and subsequent Ca2+ entry. Further, genetic deletion of TRPV4 diminished flow or shear stress-induced vasodilation in mouse carotid arteries (44, 69). TRPV4-mediated Ca2+ in response to shear or acetylcholine triggers activation of endothelial nitric oxide synthase (eNOS) and NO production which induces vasodilation through the activation of cyclic GMP/PKG/MLCP cascade in smooth muscles. Calcium influx via TRPV4 channels was also demonstrated to activate Ca2+-activated potassium channels (KCa) [Intermediate (IK) and small (SK)] and EC membrane hyperpolarization followed by deactivation of SMC voltage-dependent Ca2+ channels (VDCCs) leading to vasodilation (21). However, the coupling of TRPV4 to eNOS or IK/SK channels is determined by differential spatial organization of signaling elements in resistance arteries (90). Once TRPV4 channels are activated with agonists or physical stimuli, they increase the influx of calcium and release nitric oxide, which causes vasodilation (60). The vasodilating functions of TRPV4 may make this channel a good molecular candidate in treating vascular-related diseases.

TRPV4 and Endothelial Mechanotransduction

We were first to report that TRPV4 is activated in response to cyclic stretch and that TRPV4-mediated calcium influx is necessary for cyclic strain-induced reorientation of EC (113). Additionally, we demonstrated that mechanical force applied through β1 integrins causes TRPV4-dependent ultra-rapid calcium influx (within 4 ms) in EC (75). We demonstrated that TRPV4 is confined to focal adhesions and is activated by mechanical force transmission through CD98, a β1 integrin cytoplasmic tail binding protein (75). Deletion of the distal portion of the β1 integrin cytoplasmic domain that binds to CD98 or direct application of force to CD98, activated calcium signals within focal adhesions, confirming TRPV4 as a mechanosensory in EC. Then, we analyzed the downstream signaling pathway and found that TRPV4 controls the cyclic strain-induced reorientation of EC by activating PI3K, which in turn controls cytoskeletal reorientation and angiogenesis by modulating Rho/Rac (Figure 4B). Rho and Rac were also implicated in vascular permeability by regulating the E-cadherin junctions which were reviewed in detail by Wojciak-Stothard et al. (132). Additionally, shear stress activates TRPV4, which causes Ca2+ influx and triggers additional Ca2+-dependent IP3 receptors and this process is regulated by Rho/Rac signaling and causes vasodilation in ECs (13, 31, 46, 130, 143). Shear stress causes TRPV4 channels to leave adherens junctions and decreases their interaction with β-catenin, which is regulated by FAK and α5β1 integrins (12). Importantly, deletion or downregulation of TRPV4 reveals the crucial role for TRPV4 mechanotransduction in regulating endothelial physiology. We showed that the lack of or downregulation of TRPV4 causes aberrant mechanosensitivity toward matrix rigidity in EC through the increase of Rho/Rho kinase activity, which promotes cell spreading, migration, and pathological angiogenesis (5). Moreover, we discovered that the absence of TRPV4 causes cyclin-ERK-pathway to be upregulated, in turn promoting endothelial proliferation (114). In vivo, TRPV4KO mice displayed accelerated tumor growth, aberrant angiogenesis, vascular instability, and metastasis in a subcutaneously implanted syngeneic tumor model. Additionally, pharmacological TRPV4 activation restored aberrant vasculature and enhanced cancer treatment (5). The endothelial-specific deletion of TRPV4 recapitulated similar tumor phenotypes in mice with global TRPV4KO (56, 57). Since mechanical stresses such as cyclic strain, shear stress, and ECM stiffness can activate TRPV4, which subsequently drives actin remodeling, it is conceivable that TRPV4 may play a role in conditions such as tumor, diabetic retinopathy, and various cardiovascular diseases that involve pathological angiogenesis.

TRPV4 in Retinal Endothelial cells

TRPV4 was shown to express in primary retinal microvascular endothelial cells (RMECs) and that incubation with 25 mM d-glucose reduced TRPV4 expression and calcium influx in response to 4αPDD and GSK1016790A (81). Further, in vivo in streptozotocin-induced diabetic rats, TRPV4 expression in the retinal vascular endothelium was reduced in comparison with that in age-matched controls (81). These findings suggested that hyperglycemia and diabetes reduce functional expression of TRPV4 in retinal microvascular EC and may contribute to diabetes induced retinopathy. The same group, however, demonstrated that blockade of TRPV4 channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. Moreover, TRPV4 formed functional heteromeric channel complexes with TRPV1 in RMECs and that inhibition of either TRPV4 or TRPV1 reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the oxygen-induced retinopathy (OIR) mouse model (89). In contrast, using TRPV4KO mice, Cappelli et al. demonstrated that the absence of TRPV4 increases OIR-induced neovascularization compared to WT suggesting that TRPV4 regulates pathological but not physiological angiogenesis (17).

TRPV4 in Tumor Endothelial Cells

TRPV4 was demonstrated to regulate EC reorientation in response to cyclic strain via integrin-to-integrin signaling (113). TRPV4 expression levels were demonstrated to be lower in tumor endothelial cells (TEC) that exhibited aberrant mechanosensitivity, leading to increased cell proliferation, migration, and abnormal angiogenesis (5, 115). Mechanistically, TEC failed to reorient their actin cytoskeleton in response to uniaxial cyclic strain like TRPV4 siRNA-treated EC, suggesting that TEC exhibit a TRPV4 knockdown phenotype (37, 113). Downregulation or deletion of TRPV4 causes abnormal Rho/Rho Kinase activity, which imparts abnormal mechanosensing toward matrix stiffness, increased migration, failed reorientation in response to stretch, and ability to form and stabilize tubes on Matrigel. In fact, pretreatment of TEC with Rho kinase inhibitor Y27632 normalized abnormal mechanosensing and aberrant angiogenesis (114, 115). Interestingly, it has also been demonstrated that the activation of ERK1/2 signaling in TEC or TRPV4 KO EC correlated with increased cell proliferation and migration (115).

Tumor Microenvironment: Extracellular Vesicles

The TME attracts EC and pericytes that are essential for tumor angiogenesis, thereby acting as a driving force behind this process. EC undergo phenotypic alterations and acquire aberrant characteristics after being recruited to the TME. In many different carcinomas, TEC are weakly linked, asymmetrical, lose complete pericyte coverage, and grow on top of one another. Tumor vasculature differs from healthy ones in that it is dilated, tortuous, hyperpermeable, has enlarged cell-cell junctions, and has inadequate basement membrane covering. Collectively, these aberrant characteristics of tumor vessels produce heterogeneous blood flow and restrict nutritional supplementation inside the tumor volume (45, 124), creating a barrier to effective anticancer drug delivery (33) while also acting as a pathway for tumor cell spread. An imbalance between pro- and antiangiogenic factors was demonstrated to be the cause of the irregular features of the tumor vasculature (49). The cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), pericytes, EC, immune cells, and tumor cells are just a few of the many different cell types that make up the highly complex TME (Figure 5A; Table 2) (70, 126). These cells actively secrete growth factors and ECM that aid in tumorigenesis, angiogenesis, and metastasis. Several soluble factors, including VEGF, PDGF, tumor necrosis factor-α (TNF-α), and epidermal growth factor (EGF), are secreted by the cells in the TME. Calcium influx through ion channels controls the release of these substances, which in turn, modulates tumor growth. One of these ion channels, TRPV4, governs cell proliferation, differentiation, apoptosis, and migration, impacting the initiation and development of tumors. Importantly, TRPV4 expression is significantly reduced in TEC, although it is unknown how tumors and TME control TRPV4 in TEC (5, 17, 114, 115).

Figure 5.

Figure 5

Regulation of tumor angiogenesis by tumor microenvironment (TME) secreted extracellular vesicle (EVs) mediated downregulation of TRPV4. (A) Schematic representation of heterogenous cell population of tumor microenvironment. (B) Schematic of proposed molecular mechanisms of TRPV4 downregulation and mediated abnormal angiogenesis by EVs. EVs secreted by tumors (t-EVs) released into the TME act on endothelial cells, leading to the downregulation of TRPV4 channels, posttranslationally. TRPV4 downregulation, subsequently, induces Rho/Rho kinase-mediated YAP nuclear translocation leading to VEGFR2 translocation from the perinuclear region to the plasma membrane through unknown mechanisms (dashed line). YAP commonly binds to transcription factors such as TEAD, that initiate gene expression involved in cellular growth and proliferation; however, the mechanism by which YAP activates VEGFR2 translocation is unknown. Elevated VEGFR2 levels at the plasma membrane result in increased VEGFR2 activation by VEGF, resulting in EC proliferation, migration, and eventually, enhanced angiogenesis. Figure created with Biorender.com.

Table 2.

Immune Components and Their Functions in the Tumor Microenvironment (TME)

Immune components of TME Functions Stimulatory cytokines in TME
Macrophages Immunoregulation, matrix remodeling, angiogenesis IL10, TNFα
Dendritic cells Migrate to secondary lymphoid organs IL6, IL12, IFN type 1,TGFβ, VEGF
Neutrophils Release of reactive oxygen species, support angiogenesis, cancer cell migration and invasion, immune surveillance, and metastasis TGFβ
Natural killer cells Promote antibody-dependent cellular cytotoxicity, enhanced killing TGFβ, IL10, PGE2
Myeloid-derived suppressor cells Suppress innate and adaptive immune responses CCL2, CCL7, HIf1α, CXCL1

The numerous cell types that make up the TME communicate with one another in different ways. Extracellular vesicles (EVs) are one form of communication. EVs are tiny vesicles made up of microvesicles (100–1000 nm), exosomes (30–150 nm), and apoptotic bodies (up to 5000 nm), which include proteins, DNA, RNA, and microRNAs (miRNAs) (25, 144). Multivesicular bodies (MVBs), which fuse to the cell’s plasma membrane and are discharged into the extracellular environment and subsequently into the circulation, are precursors to the formation of exosomes. The Rab GTPase family and the endosomal sorting complex required for transport (ESCRT), both regulate this process (134). After that, EVs can be taken up by the recipient cells by endocytosis. Less is known about how microvesicles are created, but they do entail the plasma membrane budding outward (26). When a cell within the TME releases an EV, recipient cells take in its cargo and carry out downstream signaling (99).

The function of EVs in the development of cancer has recently gained attention. It is interesting to note that tumor-derived EVs (t-EVs) influence nontumor cells, recruiting them as “helpers” in tumor maintenance. t-EVs have recently come to light as crucial mediators of angiogenesis. After Skog et al. discovered that the angiogenic proteins such as tissue inhibitor of metalloproteinase-2 (TIMP-2), interleukin-6 (IL-6), IL-8, TIMP-1, VEGF, and angiogenin were abundant in glioblastoma-derived EVs (107), many studies have attempted to reveal additional mechanisms of EV-mediated angiogenesis. Adenosine transported by t-EVs binding to A2B receptors has been suggested as one promotor of tumor angiogenesis (71). Tspan8, a protein belonging to the tetraspanin family, has also been discovered in tumor-derived EVs, and when interacts with EC, it promotes tumor angiogenesis (86). EC can take up miR-181b-5p-containing EVs from esophageal squamous cell carcinoma, which is thought to enhance angiogenesis by targeting phosphatase and tensin homolog (PTEN) and PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) (122). Epidermal growth factor receptor (EGFR), Ephrin type-B receptor 2 (EPHB2) (103), miR-148a (120), miR-9–5p, piRNA-823 (65), and early growth response 1 (Egr-1) (139) are additional proangiogenic substances trafficked by t-EVs. Furthermore, the differentiation of fibroblasts into myofibroblasts, a contractile cell involved in fibrosis and inflammatory angiogenesis, is also triggered by t-EVs that express transforming growth factor beta (TGFβ) (125). These findings highlight the importance of t-EVs in tumor angiogenesis and implicate t-EVs as potential therapeutic targets in cancer therapy.

Another important role of t-EVs is shown in the lung tissue, where they promote vascular leakage, while preparing the premetastatic niche, an area where tumor cells will eventually metastasize to (94). Moreover, miR-25–3p-containing t-EVs control EC expression of VEGFR2, which promotes vascular leakage and subsequent metastasis (142). EVs produced from glioblastomas can express Semaphorin3A (Sema3A), another protein that affects vascular permeability, particularly in nervous tissue. Accordingly, brain endothelial permeability significantly increases when Sema3A is expressed in EVs (118). Similarly, clear cell renal carcinoma (ccRCC)-derived EVs and surrounding tissues contain azurocidin (AZU1), another pro-permeability factor. Importantly ccRCC patients’ serum, displayed elevated levels of AZU1-expressed EVs and higher endothelial permeability (55). Endothelial permeability causes vascular leakage, which reduces drug perfusion to the tumor and increases metastasis. As a result, the main effect of reducing vascular permeability is to make chemotherapy more effective and decrease tumor growth.

The immune system plays an important role in the prevention of tumor development. The primary cell type involved in the destruction of tumor cells is natural killer (NK) cells, which is a subset of T cells and a component of the innate immune system. Tumors that are enriched in NK cells have a better prognosis, according to patient biopsies (23). Several mechanisms are used by NK cells to attack tumor cells. Tumor cells may undergo apoptosis when exposed to Apo2 ligand (Apo-2L), a member of the tumor necrosis factor (TNF) family of proteins. It has been demonstrated that NK cells express Apo-2L and trigger the apoptosis of tumor cells when IL-2 is activated (140). A portion of T cells that are exposed to antigens develop into memory T cells, which can respond to a previously encountered antigen. This is a feature of the adaptive immune system. Cancer cells must overcome the barriers of both the innate and adaptive immune systems to grow and eventually metastasize. T cell dysregulation can happen when T cells are overstimulated with the same antigen, allowing tumor cells to proliferate unchecked (135). Cancer cells can suppress the immuno-logical response and trick the immune system by secreting EVs in the TME, a process known as “immune evasion.” For instance, EVs expressing programmed cell death ligand-1 (PD-L1), secreted by glioblastomas, drastically lower T cell activation (100). Similarly, the presence of PD-L1 and TGFβ in EVs produced from HER2-positive breast cancer cells also resulted in resistance to antibody-dependent cell cytotoxicity (ADCC) (73). Furthermore, it has been demonstrated that t-EVs suppress NK cell proliferation that is mediated by the cytokine IL-2 (22). Based on our current understanding of t-EVs’ involvement in immune system evasion, further investigations should focus on exploiting them for their usage in cancer therapy (112).

TRPV4 Mechanotransduction in Tumor Angiogenesis: Role of EVs

Although mechanical signaling in normal endothelial cells (NEC) has long been known, until recently, little was known about mechanotransduction in tumor vasculature. First, Ghosh et al. showed that TEC form aberrant vessels in 2D and 3D angiogenesis experiments appear to lose their capacity to sense physical cues (37). TEC failed to reorient in response to cyclic strain and had abnormal activation of GTPase Rho/Rock signaling. The ROCK inhibitor, Y27632 was able to reverse the aberrant behavior of TEC (37). This study has demonstrated that abnormal behavior of TEC is caused by aberrant activation of Rho signaling; however, the upstream mechanosensor that drives this abnormal behavior has not yet been explored. Thodeti et al. first showed that TRPV4 functions as a mechanosensor in EC. TRPV4 siRNA-knocked down EC did not reorient in response to the cyclic stretch, similar to TEC, suggesting that TRPV4 regulates the normal mechanosensory behavior of EC. Importantly, TRPV4 activates β1-integrin-PI3K-β1-integrin signaling to enable cyclic stretch-induced EC reorientation (113). Recent studies showed decreased functional expression of TRPV4 in TEC. TEC showed abnormal mechanosensing, migration, and proliferation compared to NEC (5, 17, 114), which is mediated by high basal Rho activity due to deletion of TRPV4. Moreover, in vivo syngeneic Lewis lung carcinoma (LLC) tumors from TRPV4 deficient mice showed increased tumor growth compared to WT mice. In addition, immunostaining of tumor vasculature with cluster of differentiation 31 (CD31) and neuron-glial antigen 2 (NG2) and injection of fluorescent dextran demonstrated that TRPV4KO tumor vessels are hyper-dilated, and leaky compared to WT tumors, suggesting that the absence of TRPV4 increases tumor angiogenesis, resulting in aberrant and hyperpermeable vessels (5, 17, 114). Importantly, the aberrant behavior of TEC in vitro was reversed by overexpressing TRPV4, or by pharmacologically activating TRPV4 with GSK1016790A (5). GSK1016790A-treated mice exhibited improved vascular stability by normalizing the leaky vessels, which was demonstrated by increased pericyte coverage of tumor vessels. This suggests that agonist-induced increase in TRPV4 expression is beneficial for normalizing the vasculature in tumors. In addition, mice treated with the anticancer drug, cisplatin, together with pretreatment of TRPV4 agonist GSK1016790A, but not alone, normalized tumor vasculature and reduced tumor volume, suggesting that TRPV4 activation normalized abnormal vasculature and enhanced cisplatin delivery throughout the tumor, resulting in decreased tumor growth (4, 115). A different study found that breast TEC expressed more TRPV4, and activating TRPV4 with arachidonic acid and 4-αPDD promoted migration of breast TEC as compared to normal breast EC (32). Further, these authors showed that cytoskeletal rearrangement that resulted from TRPV4 activation with arachidonic acid, increased TRPV4 membrane translocation. However, the in vivo significance of TRPV4 in tumor angiogenesis was not examined in this study.

Although studies have shown that downregulation of TRPV4 is critical for tumor angiogenesis, the molecular mechanism by which a decrease in TRPV4 functional expression promotes tumor angiogenesis remains unknown. Endothelial activation, proliferation, migration, tube formation, and maturation are the major steps of angiogenesis. Thoppil et al. showed that, in comparison to NEC, TEC with lower TRPV4 expression exhibits higher proliferation. Moreover, TEC show elevated expression of proliferative genes crucial for the G1/S phase of the cell cycle as well as significant basal ERK1/2 phosphorylation. Importantly, pharmacological activation of TRPV4 with GSK1016790A reduced TEC proliferation without affecting the NEC (114). Additionally, this decline in proliferation of TEC was associated with a decline in the expression of genes that promote cell cycle progression. Notably, pharmacological activation of TRPV4 in vivo decreased TEC proliferation without affecting the tumor cells themselves (114). Recent studies showed that TRPV4 knockdown induced activation of VEGFR2 via mechanotranscriptional Rho/Rho Kinase/YAP pathway (3, 38, 57) (Figure 5B). Further, increased VEGFR2-positive vessels were demonstrated in LLC tumors induced in TRPV4KO mice. Furthermore, LLC tumors implanted in endothelial-specific TRPV4KO mice (TRPV4ECKO) exhibited enhanced tumor angiogenesis and lung metastasis compared to WT (TRPV4lox/lox) mice. It has been shown that elevated VEGFR2, activation of ERK, and matrix metalloprotease-9 (MMP-9) are the molecular mechanisms downstream of endothelial deletion of TRPV4 (56).

Although these studies showed that TRPV4 deletion/downregulation in EC increased tumor angiogenesis and progression, it is unknown how TME downregulates TRPV4 in EC. To study this, we repeatedly subjected NEC to lung carcinoma (A549) cell-conditioned media to mimic the TME. Normal human microvascular EC (HMEC-1) treated with tumor cell conditioned media showed elevated expression of tumor endothelial marker 8 (TEM8) and aberrant tube formation compared to untreated EC suggesting that they underwent a transformation into tumor-like EC after exposure to TME.

Further, these EC showed higher VEGFR2 activation. Importantly, TRPV4 expression was decreased in NEC exposed to tumor-conditioned medium, indicating that TME may transform NEC to a TEC-like phenotypic via TRPV4 reduction. Next, we discovered that tumor cell conditioned media treated with the exosome inhibitor, GW4869, prevented the transformation of TEC, indicating that t-EVs transform ECs via the downregulation of TRPV4, which we confirmed by exposing naive ECs to t-EVs isolated from conditioned media (38, 39). Finally, TRPV4 downregulation by t-EVs resulted in subsequent activation of YAP/VEGFR2 and aberrant angiogenesis (38) (Figure 5B).

Conclusions and Perspectives

Angiogenesis is a fundamental requirement for the growth of solid tumor tissue. Several strategies have been developed to target tumor-associated angiogenesis aimed at improving cancer therapy and reducing tumor growth and metastasis. These include new drugs that have been identified to act against VEGF and the VEGFR family, extracellular vesicle-mediated regulation of tumor angiogenesis, and identification of mechanisms that provide resistance to antiangiogenic drugs (Figure 6). Even though, antiangiogenic therapy remains the first line of therapy to date, it has failed to improve overall survival. Similarly, neovascularization has been a critical factor in AMD and PR that leads to blindness. Although anti-VEGF therapy adopted from the tumor studies has shown very good promise, side effects such as retinal vasculitis and intraocular inflammation were reported in AMD or PR patients (Figure 2). Therefore, there is an urgent need to identify novel mechanistic strategies to target pathological angiogenesis. Activation of TRPV4 supports retinal normalization and will also have additional beneficial effects in regulating intraocular pressure (IOP) and outflow facility. A recent study by Patel et al. demonstrated that the activation of TRPV4 showed significantly reduced IOP with improved outflow facility in mice. In addition, the deletion of TRPV4 in trabecular meshwork (TM) tissue (TRPV4−/−TM) showed significant increase in IOP. Together these data conclude, the critical role of TRPV4TM in regulating IOP (93). Therefore, we propose external application of TRPV4 activators as eye drops could induce normalization of retinal vasculature with additional beneficial effects of reduced IOP and improved outflow facility, which may altogether aid in avoiding repeated injections of anti-VEGF drugs into the eye (Figure 2). Normalization of the tumor vasculature has been identified as an alternative mechanism for antiangiogenesis in cancer therapy, which facilitates uniform blood flow throughout the tumor tissue and subsequent improvement of drug delivery. Interestingly, vascular normalization techniques still employ anti-VEGF and anti-PDGF therapies, to which tumors show resistance and other adverse side effects. We have previously demonstrated that endothelial TRPV4 is a critical modulator of vascular integrity, tumor angiogenesis, and metastasis and that pharmacological activation of TRPV4 normalizes tumor vasculature thereby improving cancer therapy (Figure 6). Hence, unlike targeting VEGF signaling, which is critical for basal vascular function, targeting endothelial TRPV4 would offer a potential therapeutic avenue for vascular normalization and cancer therapy.

Figure 6.

Figure 6

Proposed mechanisms by which TRPV4 acts as a VEGF-independent target for vascular normalization. (A) Conventionally, aberrant VEGF/VEGFR2 signaling appears to be responsible for tumor angiogenesis, and targeting this signaling pathway using anti-VEGF drugs would inhibit tumor angiogenesis and tumor growth. However, tumor vasculature develops resistance or finds alternative pathways such as FGF to induce angiogenesis in the presence of anti-VEGF molecules i.e. escape angiogenesis (top). TRPV4 activation in TEC induces vascular normalization, independent of VEGF/FGF signaling via modulation of Rho/MMP-9 signaling and restores vascular integrity (bottom). (B) TRPV4-dependent antiangiogenic therapy and vascular normalization (bottom). TRPV4 activation by GSK1 normalizes tumor microenvironment and tumor vasculature and improves blood flow (middle). TRPV4 activation together with chemo- or radiotherapy improves drug delivery and/or tumor cell death resulting in the reduction of tumor growth and metastasis (right). Abbreviations: TRPV4, transient receptor potential vanilloid type 4; EC, endothelial cell; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; FGF, fibroblast growth factor; NEC, normal endothelial cell; TEC, tumor endothelial cell; MMP9, matrix metalloprotease-9; GSK1, GSK 1016790A; YAP, yes-associated protein. Figure created with Biorender.com.

Didactic Synopsis.

Major teaching points

  • Angiogenesis is the process of formation of new blood vessels from the existing vessels.

  • Angiogenesis is critical for maintaining normal cardiovascular function.

  • Excessive or inadequate angiogenesis can lead to pathologies such as cancer, proliferative retinopathy (excessive), or ischemic heart disease (myocardial infarction).

  • VEGF/VEGFR2 pathway is the major mediator of angiogenesis. Therefore, antiangiogenic therapies are focused on targeting VEGF signaling; however, they have met with limited success.

  • Vascular normalization is an alternative approach for anti-angiogenic therapies, but the targets are still the soluble growth factors such as VEGF, FGF, and PDGF.

  • TRPV4 is mechanosensitive ion channel in endothelial cells and TRPV4 mechanotransduction appears to be dysregulated in pathological angiogenesis.

  • Targeting TRPV4 could be a novel VEGF-independent alternative for antiangiogenic and vascular normalization approaches for improved therapy.

Funding

This work was supported by the National Institutes of Health (NIH) (R01HL119705, R01HL148585, and R15CA202847; American Heart Association (AHA) Transformational Project Award # 971237 to CKT; and R01AI144115 to SP) and T32HL134625 (BG).

Footnotes

Credit Authorship Contribution Statement

Venkatesh Katari and Charles K. Thodeti conceived the project. Venkatesh Katari, Kesha Dalal, Ravi K. Adapala, Brianna D. Guarino, Narendrababu Kondapalli, Sailaja Paruchuri, and Charles K. Thodeti wrote and revised the manuscript.

Declaration of Competing Interest

None declared.

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