Endothelial Cell Motility Research Articles

Raised SPINK1 levels play a role in angiogenesis and the transendothelial migration of ALL cells

The present study was designed to assess whether raised Serine protease inhibitor Kazal type 1 (SPINK1) expressions modulates angiogenesis. Human umbilical vein endothelial cells (HUVECs) exposed to SPINK1 were noted to exhibit raised expressions of interleukin-8 (IL-8) as well as VCAM-1 and ICAM-1 cell adhesion molecules in a dose-dependent manner. In co-culture system of HUVECs and Acute lymphoblastic leukemia (ALL) cells, SPINK1 exposure also resulted in enhanced endothelial cell motility and ALL cells trans-endothelial migration. High concentrations of SPINK1 caused in vitro cellular reorganization into tubes in Matrigel-cultured HUVECs and induced in vivo vascularization and brain infiltration of NOD/SCID ALL model mice. The further transcriptomic analysis indicated that SPINK1 treatment altered several biological processes of endothelial cells and led to activation of the MAPK pathway. This study is the first to determine the neovascularization effects of raised SPINK1.

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs.

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice.

Lymphatic vascular development is regulated by well-characterized signaling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity, and morphogenesis. Canonical and non-canonical WNT signaling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signaling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signaling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signaling during the formation of lymphatic vascular networks.

An Essential NRP1-Mediated Role for Tagln2 in Gastric Cancer Angiogenesis.

Knowledge about the precise biological role and underlying mechanism of Tagln2 in tumor progression is relatively limited, especially in angiogenesis focused on tumor derived endothelial cells (ECs) has rarely been reported. Here, the function, molecular mechanism and potential clinical value of Tagln2 in gastric cancer (GC) angiogenesis were investigated. GC tissue microarrays were used to assess the expression of Tagln2 in ECs. The relationships between expression and clinicopathological features were analyzed to evaluate the clinical value of Tagln2. Gain- and loss-of-function approaches were performed in ECs to investigate the functions of Tagln2 in angiogenesis. A combination of angiogenesis antibody array, RNA-Seq analyses and a series of in vitro experiments were performed to reveal the proangiogenic mechanism mediated by NRP1. Immunohistochemistry performed on an independent tissue chip (n=75) revealed significant upregulation of Tagln2 in tumor-derived ECs which were specifically immunolabeled with CD34. Additionally, high Tagln2 levels correlated significantly with the presence of lymph node as well as distant metastases. Gain- and loss-of-function approaches highlighted the function of Tagln2 in promoting EC proliferation, motility, and capillary-like tube formation and in reducing apoptosis. Tagln2 upregulation led to significantly increased mRNA and protein levels of NRP1 and subsequently activated the NRP1/VEGFR2 and downstream MAPK signaling pathways. These data indicate the importance of Tagln2 in angiogenesis, as a potential therapeutic target, and as a candidate prognostic marker in GC.

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells.

Single-cell motility is spatially heterogeneous and driven by metabolic energy. Directly linking cell motility to cell metabolism is technically challenging but biologically important. Here, we use single-cell metabolic imaging to measure glycolysis in individual endothelial cells with genetically encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. We show that cellular glycolysis fuels endothelial activation, migration and contraction and that sites of high lactate production colocalize with active cytoskeletal remodelling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Our data indicate that Rho-GTPase signalling coordinates energy metabolism with cytoskeleton remodelling to regulate endothelial cell motility.

Endothelial metabolism going single.

Endothelial cell migration is indispensable for (tumour) angiogenesis and is fuelled by glycolysis. The metabolic underpinnings have been only partially unravelled to date, partly because of the lack of appropriate tools to analyse metabolic flux at the single-cell level. In this issue, Wu et al. introduce a novel imaging assay to capture glycolytic flux during motion in single endothelial cells. With this new tool, the authors uncovered a glycolysis-driven cytoskeletal remodelling apparatus that propels endothelial cell motility.

CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein-coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

MiR-203 Targets to the 3'-UTR of SLUG to Suppress Cerebral Infarction-Induced Endothelial Cell Growth and Motility.

Cerebral infarction is one of the leading causes of death worldwide, in which angiogenesis plays a critical role. On the other hand, accumulating evidence has demonstrated that microRNAs (miRNAs) function as key modulators in the formation and progression of cerebral infarction. However, the molecular mechanisms of miRNAs underlying cerebral infarction-associated angiogenesis remain unclear. In the present study, we indicated that the expression of miR-203 was significantly downregulated in serum samples derived from patients with cerebral infarction and in mice brain samples following middle cerebral artery occlusion (MCAO) compared with healthy controls. In vitro, the expression of miR-203 was obviously downregulated in hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs). Functionally, ectopic expression of miR-203 drastically suppressed HUVEC proliferation, invasion, and migration. In addition, SLUG, a zinc finger transcriptional repressor, was identified as a direct target of miR-203 and was negatively correlated with miR-203 expression in MCAO mice and in hypoxia-induced HUVECs. Furthermore, overexpression of SLUG reversed the inhibitory effect of miR-203 on proliferation, invasion, and migration abilities of HUVECs. Taken together, our research provides a novel insight of the miR-203-SLUG axis into cerebral infarction-associated endothelial behaviors and may offer a powerful therapeutic target of cerebral ischemia.

Effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism

Objective: To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism. Methods: The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased (P<0.05 or P<0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group (P<0.01) and hypoxia+ BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly (P<0.05 or P<0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions: BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells

The tight junction protein Claudin-5 limits endothelial cell motility.

Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells. In vitro and in vivo studies demonstrate that loss of claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there isno information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

Elucidating Angiogenesis: The Role of Basement Membrane Proteolysis and Endothelial Cell Motility and Proliferation

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RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis

Polarised targeting of diverse mRNAs to cellular protrusions is a hallmark of cell migration. Although a widespread phenomenon, definitive functions for endogenous targeted mRNAs and their relevance to modulation of in vivo tissue dynamics remain elusive. Here, using single‐molecule analysis, gene editing and zebrafish live‐cell imaging, we report that mRNA polarisation acts as a molecular compass that orients motile cell polarity and spatially directs tissue movement. Clustering of protrusion‐derived RNAseq datasets defined a core 192‐nt localisation element underpinning precise mRNA targeting to sites of filopodia formation. Such targeting of the small GTPase RAB13 generated tight spatial coupling of mRNA localisation, translation and protein activity, achieving precise subcellular compartmentalisation of RAB13 protein function to create a polarised domain of filopodia extension. Consequently, genomic excision of this localisation element and perturbation of RAB13 mRNA targeting—but not translation—depolarised filopodia dynamics in motile endothelial cells and induced mispatterning of blood vessels in zebrafish. Hence, mRNA polarisation, not expression, is the primary determinant of the site of RAB13 action, preventing ectopic functionality at inappropriate subcellular loci and orienting tissue morphogenesis.

Endothelial response boosted by platelet lysate: The involvement of calcium toolkit

Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca2+) signals play a central role in the control of endothelial cell survival, proliferation, motility, and differentiation. We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i.

Abstract 1489: Exosomes are key players in nicotine-induced angiogenesis

Abstract Nicotine, an addictive component of cigarette smoke and tobacco, have been shown to promote cancer pathogenesis. Angiogenesis is an important hallmark of cancer that sustains the supply of nutrients and oxygen to the fast proliferating cancer cells through the formation of new blood vessels. The goal of present study was to examine the direct and indirect effect of nicotine on endothelial cell growth, their migration and invasion, and capillary forming ability. We treated the human umbilical vein endothelial cells with two doses of nicotine within physiological range of smokers (200nM and 400nM) and examined the effect on their growth. In parallel, we treated the prostate cancer cells with similar doses of nicotine or vehicle for 8 h and subsequently replaced the media. After next 48 h, conditioned media (CM) was collected from vehicle- (Veh-CM) and nicotine- (Nic-CM) treated prostate cancer cells and used for the treatment of endothelial cells. The growth of endothelial cells increased by ~19 % and ~22 % upon direct treatment of nicotine at 200nM and 400nM doses, respectively. However, a far greater increase was recorded (~128% and ~ 146%, respectively), when endothelial cells were treated with the conditioned media derived from nicotine-treated cancer cells at 200nM and 400nM doses. In next of experiments, we fractionated Veh-CM and Nic-CM (200nM dose) into soluble and exosomal fractions and examined their effect on endothelial cells. We observed that exosomal fraction was more potent (~89%) in inducing endothelial cell growth as compared to the soluble fraction (~46%). In addition, we also observed significant greater increase in motility, invasion and capillary-forming ability of endothelial cells that were pre-treated with Nic-Exo as compared to those treated with Veh-Exo. Studies are currently ongoing to examine the comparative efficacy of nicotine-treated soluble and exosomal factions in potentiating motility, invasion and capillary formation and define the underlying molecular mechanism(s). Our findings thus support a novel pathogenic role of nicotine in cancer where exosomes act as key players in promoting angiogenesis. Citation Format: Shubhangi Singh, Sirin Saranyutanon, Mohammad Aslam Khan, Sanjeev Kumar Srivastava, Seema Singh, Ajay P. Singh. Exosomes are key players in nicotine-induced angiogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1489.

P28 Bacterial Peptide, as an Anticancer Agent.

Cancer remains a major cause of morbidity and mortality irrespective of the type of conventional chemotherapy. Therefore, there is an urgent need for new and effective anticancer therapeutic agents. Bacterial proteins and their derivative peptides appear as a promising approach for cancer treatment. Several, including an amphipathic, α-helical, 28-amino acid peptide derived from azurin, a 128-amino acid copper-containing redox protein secreted from Pseudomonas aeruginosa, show clinical promise in the treatment of adult and pediatric solid tumors. The peptide, p28, is a post-translational, multi-target anticancer agent that preferentially enters a wide variety of solid tumor cells. Mechanistically, after entry, p28 has two major avenues of action. It binds to both wild-type and mutant p53 protein, inhibiting constitutional morphogenic protein 1 (Cop1)-mediated ubiquitination and proteasomal degradation of p53. This results in increased levels of p53, which induce cell-cycle arrest at G2/M and an eventual apoptosis that results in tumor cell shrinkage and death. In addition, p28 also preferentially enters nascent endothelial cells and decreases the phosphorylation of FAK and Akt inhibiting endothelial cell motility and migration. Here, we review the current basic and clinical evidence suggesting the potential of p28 as a cancer therapeutic peptide.

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ) inhibits cancer cell metastasis behavior of TNBC via suppressing EMT and VEGF

Triple-negative breast cancer (TNBC) is highly metastatic and lacks effective therapeutic targets among several subtypes of breast cancer. Cancer metastasis promotes the malignancy of TNBC and is closely related to the poor prognosis of the TNBC patients. We aim to explore novel agents that effectively inhibit cancer metastasis to treat TNBC. In our study, 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ), a CA-4 analogue, could inhibit cell motility and invasion in MDA-MB-231 cells, and the mechanism is closely associated to the inhibition of epithelial-to-mesenchymal transition (EMT). Meanwhile, SQ significantly inhibited the expression and secretion of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells. Moreover, the conditioned medium from SQ-treated MDA-MB-231 cells significantly inhibited the motility and invasion of human umbilical vein endothelial cells (HUVECs), which was correlated with the inhibition of EMT process in HUVECs. In addition, exogenous application of VEGF reversed the occurrence of EMT in HUVECs which stimulated by conditioned medium from SQ-treated cells. Furthermore, SQ inhibited vasculogenic mimicry (VM) formation in MDA-MB-231 cells, which was associated with VE-cadherin and EphA2 down-regulation. This study indicates that SQ inhibits MDA-MB-231 cell metastasis through suppressing EMT and VEGF, thereby implicating this compound might be a potential therapeutic agent against metastatic TNBC.

Targeting Soluble Epoxide Hydrolase with TPPU Alleviates Irradiation‐Induced Hyposalivation in Mice via Preventing Apoptosis and Microcirculation Disturbance

AbstractIR irradiation‐induced dysfunction of the salivary gland and hyposalivation are one of the main complications of radiotherapy treatment for malignant head and neck tumors, and its mechanisms are not fully understood. IR‐induced microcirculation disturbance and apoptosis is one of the most important mechanisms. Most recent studies have focused on the regenerative effects of epoxyeicosatrienoic acids (EETs) through the process of increased angiogenesis. This study suggests that increasing endogenous EETs by the soluble epoxide hydrolase (sEH) inhibitor TPPU can reverse dysfunctional salivary glands and hyposalivation by improving the associated microenvironment and tissue regeneration. The findings suggest that TPPU alleviates hyposalivation and histological lesions, increases angiogenesis and reduces apoptosis of acinar cells significantly in IR‐damaged mice. The results in vitro demonstrate that TPPU promotes the proliferation and inhibits apoptosis of human salivary gland (HSG) cells, and enhances the motility and angiogenesis of human umbilical vein endothelial cells (HUVECs). The results also suggest that TPPU‐induced synergy and crosstalk between acinar cells and vascular endothelial cells might contribute to angiogenesis. Together, this study proves the feasibility of targeting sEH using TPPU to promote angiogenesis and reduce apoptosis, and also indicates the potential of sEH inhibitors for functional reconstruction of IR‐induced salivary glands damage.

Therapeutic Angiogenesis Using HGF Plasmid.

Hepatocyte growth factor (HGF) is secreted from stromal and mesenchymal cells, and its receptor cMet is expressed on various types of cells such as smooth muscle cells, fibroblast, and endothelial cells. HGF stimulates epithelial and endothelial cell proliferation, motility, and morphogenesis in a paracrine and autocrine manner, organizing multistep of angiogenesis in many organs. In addition, HGF is recognized as a potent anti-inflammatory and anti-fibrotic growth factor, which has been proved in several animal studies, including neointimal hyperplasia and acute myocardial infarction model in rodent. Thus, as compared to other angiogenic growth factors, HGF exerts multiple effects on ischemic tissues, accompanied by the regression of tissue inflammation and fibrosis. These data suggest the therapeutic potential of the HGF for peripheral artery disease as it being accompanied with chronic tissue inflammation and fibrosis. In the present narrative review, the pleiotropic action of the HGF that differentiates it from other angiogenic growth factors is discussed first, and later, outcomes of the human clinical study with gene therapy are overviewed.