Lowered expression level of INSC predicts poor prognosis in patients with colon cancer.

The aim of this study was to investigate the prognostic value of inscuteable spindle orientation adaptor protein (INSC) in colon cancer (CC). Firstly, transcriptional change of INSC was analysed using the data from public databases. Next, INSC protein expression was assessed by immunohistochemistry. Its correlation with clinicopathological features and the prognostic values of patients were also investigated. Then, an INSC-based nomogram was built to predict CC prognosis. Compared to normal tissues, INSC was significantly downregulated at the transcriptional level in CC tissues. A low INSC mRNA level not only positively correlated with TNM stage (tumour-nodus-metastases), advanced T stage, and N stage, but also with the shorter 5- and 8-year overall survival (OS) and disease-specific survival. Concerning protein level, INSC downregulation was confirmed in CC samples. In terms of the correlation with N stage and 5- and 8-year OS, it was also consistent with mRNA levels. Cox regression analysis indicated that INSC protein expression was an independent prognostic factor for OS. The nomogram showed better prognostic accuracy and clinical net benefit for 5-year OS than TNM staging. Altogether, downregulation of INSC is related to inferior clinicopathological features and patient outcomes, and it may be a novel independent prognostic biomarker in CC.

[Comparative study of two elastic fixation systems in single-segment lumbar disc herniation surgery for nucleus pulposus extraction].

OBJECTIVE: To investigate the clinical application of two elastic pedicle internal fixation systems in single-segment lumbar disc herniation fenestration. METHODS: A retrospective analysis of 64 patients with lumbar intervertebral disc herniation treated by surgery from June 2019 to March 2021. According to the different elastic fixation systems placed during the operation, the patients were divided into ordinary pedicle screw elastic rod link group (elastic rod group) and a special elastic pedicle screw rigid rod fixed connection group (elastic screw group). There were 33 cases in the elastic rod group, including 18 males and 15 females, aged from 30 to 69 years old with an average of(49.18±10.23) years old;and 31 cases in the elastic screw group, including 16 males and 15 females, aged from 32 to 68 with an average of (49.81±9.24) years old. The operation time, intraoperative blood loss, postoperative wound drainage, and postoperative landing time of the two groups were recorded separately. The visual analogue scale (VAS), Japanese Orthopaedic Association (JOA) score, and Oswestry Disability Index (ODI) were compared before and 3, 12 months after operation. The height of the adjacent vertebral space on the lateral DR film before and 12 months after the operation was measured. The clinical efficacy was evaluated by Macnab standard. RESULTS: All the patients successfully completed the operation, and were followed up. The operation time, intraoperative blood loss, postoperative wound drainage and postoperative landing time in the elastic rod group were(63.73±12.01) min, (89.55±16.07) ml, (81.67±16.00) ml, (3.45±0.75) d , while in the elastic nail group was (62.96±11.54) min, (88.35±17.14) ml, (82.29±15.40) ml, (3.29±0.78) d, the difference was not statistically significant. The symptoms of low back pain and lower extremity numbness were significantly improved in all patients after operation. There was no significant difference in VAS, JOA score and ODI between the two groups before and after surgery (P>0.05). At 12 months after operation, there was no significant difference in the height of the adjacent vertebral space between the upper adjacent vertebral body and the same segment before operation(P>0.05), and there was no significant difference between the groups before and after the operation. According to Macnab criteria, the elastic rod group was excellent in 30 cases, good in 2 cases, fair in 1 case, while the elastic nail group was excellent in 29 cases, good in 2 cases, fair in 0 cases, and there was no significant difference(Z=-0.42, P=0.68). CONCLUSION: In fenestrated nucleus pulposus extraction for lumbar disc herniation, the two elastic pedicle internal fixation systems are equally effective and can be used. The elastic screw internal fixation system has certain advantages when the distance between the two vertebral bodies is short, and the elastic rod cannot be placed or is difficult to be placed, and it is more widely used.

Overexpression of TaLBD16-4D alters plant architecture and heading date in transgenic wheat.

Lateral organ boundaries domain (LBD) proteins, a class of plant-specific transcription factors with a special domain of lateral organ boundaries (LOB), play essential roles in plant growth and development. However, there is little known about the functions of these genes in wheat to date. Our previous study demonstrated that TaLBD16-4D is conducive to increasing lateral root number in wheat. In the present work, we further examined important agronomical traits of the aerial part of transgenic wheat overexpressing TaLBD16-4D. Interestingly, it was revealed that overexpressing TaLBD16-4D could lead to early heading and multiple alterations of plant architecture, including decreased plant height, increased flag leaf size and stem diameter, reduced spike length and tillering number, improved spike density and grain width, and decreased grain length. Moreover, auxin-responsive experiments demonstrated that the expression of TaLBD16-4D in wild-type (WT) wheat plants showed a significant upregulation through 2,4-D treatment. TaLBD16-4D-overexpression lines displayed a hyposensitivity to 2,4-D treatment and reduced shoot gravitropic response. The expressions of a set of auxin-responsive genes were markedly different between WT and transgenic plants. In addition, overexpressing TaLBD16-4D affected the transcript levels of flowering-related genes (TaGI, TaCO1, TaHd1, TaVRN1, TaVRN2, and TaFT1). Notably, the expression of TaGI, TaCO1, TaHd1, TaVRN1, and TaFT1 displayed significant upregulation under IAA treatment. Collectively, our observations indicated that overexpressing TaLBD16-4D could affect aerial architecture and heading time possibly though participating in the auxin pathway.

Attitudes toward COVID-19 vaccination and willingness to pay: comparison of people with and without mental disorders in China.

BACKGROUND: Acceptance and willingness to pay for the COVID-19 vaccine are unknown. AIMS: We compared attitudes toward COVID-19 vaccination in people suffering from depression or anxiety disorder and people without mental disorders, and their willingness to pay for it. METHOD: Adults with depression or anxiety disorder (n = 79) and healthy controls (n = 134) living in Chongqing, China, completed a cross-sectional study between 13 and 26 January 2021. We used a validated survey to assess eight aspects related to attitudes toward the COVID-19 vaccines. Psychiatric symptoms were assessed by the 21-item Depression, Anxiety and Stress Scale. RESULTS: Seventy-six people with depression or anxiety disorder (96.2%) and 134 healthy controls (100%) reported willingness to receive the COVID-19 vaccine. A significantly higher proportion of people with depression or anxiety disorder (64.5%) were more willing to pay for the COVID-19 vaccine than healthy controls (38.1%) (P ≤ 0.001). After multivariate adjustment, severity of depression and anxiety was significantly associated with willingness to pay for COVID-19 vaccination among psychiatric patients (P = 0.048). Non-healthcare workers (P = 0.039), health insurance (P = 0.003), living with children (P = 0.006) and internalised stigma (P = 0.002) were significant factors associated with willingness to pay for COVID-19 vaccine in healthy controls. CONCLUSIONS: To conclude, psychiatric patients in Chongqing, China, showed high acceptance and willingness to pay for the COVID-19 vaccine. Factors associated with willingness to pay for the COVID-19 vaccine differed between psychiatric patients and healthy controls.

Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang).

BACKGROUND: Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. RESULTS: In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. CONCLUSIONS: A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.

An Asia-Pacific study on healthcare workers' perceptions of, and willingness to receive, the COVID-19 vaccination.

OBJECTIVE: In the fight against COVID-19, vaccination is vital in achieving herd immunity. Many Asian countries are starting to vaccinate frontline workers; however, expedited vaccine development has led to hesitancy among the general population. We evaluated the willingness of healthcare workers to receive the COVID-19 vaccine. METHODS: From 12 to 21 December 2020, we recruited 1720 healthcare workers from 6 countries: China, India, Indonesia, Singapore, Vietnam and Bhutan. The self-administrated survey collected information on willingness to vaccinate, perception of COVID-19, vaccine concerns, COVID-19 risk profile, stigma, pro-socialness scale, and trust in health authorities. RESULTS: More than 95% of the healthcare workers surveyed were willing to vaccinate. These respondents were more likely to perceive the pandemic as severe, consider the vaccine safe, have less financial concerns, less stigmatization regarding the vaccine, higher pro-socialness mindset and trust in health authorities. A high perceived pandemic risk index, low vaccine harm index and high pro-socialness index were independent predictors in multivariable analysis. CONCLUSIONS: The majority of healthcare workers in Asia are willing to receive COVID-19 vaccination. Perceived COVID-19 susceptibility, low potential risk of vaccine harm and pro-socialness are the main drivers. These findings may help formulate vaccination strategies in other countries.

An Eight-CpG-based Methylation Classifier for Preoperative Discriminating Early and Advanced-Late Stage of Colorectal Cancer.

AIM: To develop and validate a CpG-based classifier for preoperative discrimination of early and advanced-late stage colorectal cancer (CRC). METHODS: We identified an epigenetic signature based on methylation status of multiple CpG sites (CpGs) from 372 subjects in The Cancer Genome Atlas (TCGA) CRC cohort, and an external cohort (GSE48684) with 64 subjects by LASSO regression algorithm. A classifier derived from the methylation signature was used to establish a multivariable logistic regression model to predict the advanced-late stage of CRC. A nomogram was further developed by incorporating the classifier and some independent clinical risk factors, and its performance was evaluated by discrimination and calibration analysis. The prognostic value of the classifier was determined by survival analysis. Furthermore, the diagnostic performance of several CpGs in the methylation signature was evaluated. RESULTS: The eight-CpG-based methylation signature discriminated early stage from advanced-late stage CRC, with a satisfactory AUC of more than 0.700 in both the training and validation sets. This methylation classifier was identified as an independent predictor for CRC staging. The nomogram showed favorable predictive power for preoperative staging, and the C-index reached 0.817 (95% CI: 0.753-0.881) and 0.817 (95% CI: 0.721-0.913) in another training set and validation set respectively, with good calibration. The patients stratified in the high-risk group by the methylation classifier had significantly worse survival outcome than those in the low-risk group. Combination diagnosis utilizing only four of the eight specific CpGs performed well, even in CRC patients with low CEA level or at early stage. CONCLUSIONS: Our classifier is a valuable predictive indicator that can supplement established methods for more accurate preoperative staging and also provides prognostic information for CRC patients. Besides, the combination of multiple CpGs has a high value in the diagnosis of CRC.

Restoration of GATA4 expression impedes breast cancer progression by transcriptional repression of ReLA and inhibition of NF-κB signaling.

There are increasing reports of aberrant expression of GATA4, correlated with oncogenesis and malignant progression in some solid tumors, but whether GATA4 functions as an oncogenic driver or a tumor suppressor in carcinogenesis remains controversial. Because the role and mechanism of GATA4 in breast cancer (BrCa) remain poorly understood, we focused on the expression of GATA4 in BrCa cell lines and tissues and its mechanism in breast oncogenesis. Semiquantitative real-time polymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot analysis, and immunohistochemistry were used to detect expression of GATA4 in BrCa cell lines and adjacent breast tissues. Methylation statuses of the GATA4 promoter were studied using methylation-specific PCR in BrCa cell lines.The effects of GATA4 on proliferation, invasion, and cell cycle were also analyzed. Compared with adjacent breast tissue, GATA4 expression in BrCa tissue and cell lines was obviously lower and low expression levels of GATA4 predicted poor outcome. Methylation of GATA4 occurred in almost all of BrCa cell lines . GATA4 overexpression decreased viability, invasion, migration, and epithelial-to-mesenchymal transition of MB-231 and BT549 cells, and markedly induced cell cycle arrest and apoptosis. Exogenous expression GATA4 accompanied a significant alteration of MMP2, MMP3, E-cadherin, and N-cadherin expression and induction of the caspase-8 pathway. Moreover, GATA4 could directly repress RelA (p65) transcription, reduce the nuclear phosphorylation-p65 and upregulate inhibitor kappa B expression. Altogether, GATA4 plays a tumor-suppressive role via repression of NF-κB signaling in BrCa cells. Our findings suggest that GATA4 is a potential prognostic biomarker and gene therapeutic target for human BrCa.

Myosin IIa is critical for cAMP-mediated endothelial secretion of von Willebrand factor.

Nonmuscle myosin II has been implicated in regulation of von Willebrand factor (VWF) release from endothelial Weibel-Palade bodies (WPBs), but the specific role of myosin IIa isoform is poorly defined. Here, we report that myosin IIa is expressed both in primary human endothelial cells and intact mouse vessels, essential for cyclic adenosine monophosphate (cAMP)-mediated endothelial VWF secretion. Downregulation of myosin IIa by shRNAs significantly suppressed both forskolin- and epinephrine-induced VWF secretion. Endothelium-specific myosin IIa knockout mice exhibited impaired epinephrine-stimulated VWF release, prolonged bleeding time, and thrombosis. Further study showed that in resting cells, myosin IIa deficiency disrupted the peripheral localization of Rab27-positive WPBs along stress fibers; on stimulation by cAMP agonists, myosin IIa in synergy with zyxin promotes the formation of a functional actin framework, which is derived from preexisting cortical actin filaments, around WPBs, facilitating fusion and subsequent exocytosis. In summary, our findings not only identify new functions of myosin IIa in regulation of WPB positioning and the interaction between preexisting cortical actin filaments and exocytosing vesicles before fusion but also reveal myosin IIa as a physiological regulator of endothelial VWF secretion in stress-induced hemostasis and thrombosis.

Dickkopf-Related Protein 2 is Epigenetically Inactivated and Suppresses Colorectal Cancer Growth and Tumor Metastasis by Antagonizing Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: Aberrant activation of the Wnt/ß-catenin signaling pathway plays a key role in the pathogenesis of multiple tumors including digestive cancers. Recent studies have reported that Dickkopf-related protein 2 (DKK2) is epigenetically inactivated in numerous types of cancers and that its gene products exhibit tumor-suppressive properties. However, the biological functions and underlying molecular mechanisms of DKK2 in colon carcinoma remains obscure. METHODS: We examined the expression of DKK2 in colon tumor cell lines by RT-PCR and its promoter methylation status in colon tumor cell lines and primary tumors by methylation-specific PCR (MSP). Ectopic expression of DKK2 was measured by RT-PCR prior to the other experiments. To investigate the function of DKK2, we assayed colony formation and cell proliferation, utilized flow cytometric analyses of the cell cycle and acridine orange/ethidium bromide (AO/EB) fluorescence staining for apoptosis, and examined wound healing, transwell migration and tumor growth in vivo. Western blots were used to explore the mechanisms of DKK2 in epithelial- mesenchymal transition and canonical Wnt/ß-catenin signaling. RESULTS: We show here that downregulation or silencing of DKK2 was closely associated with the hypermethylation status of its promoter and that DKK2 expression could be restored by demethylation treatment. Methylation of the DKK2 promoter was detected in nearly all tumors and tumor-adjacent tissues, but not in normal colon tissues. Ectopic expression of DKK2 in colon cell lines HCT116 and HT-29 inhibited colony formation and cell viability by inducing cell cycle G0/G1 arrest and apoptosis, and growth of stable DKK2-infected HCT116 cells in nude mice was decreased compared to controls. Furthermore, DKK2 restrained cell migration through partial reversal of epithelial-to- mesenchymal transition and also by downregulating several stem cell markers. Our data further showed that restoration of DKK2 expression resulted in downregulation of active ß-catenin and its downstream target genes. CONCLUSION: DKK2 appears to be a functional tumor suppressor regulating tumorigenesis of colorectal cancer by antagonizing Wnt/ß-catenin signaling.

Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules.

Endothelial exocytosis of Weibel-Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis.

[Cell surface adhesion molecules mediated stretch-induced HL-60 cell adhesion to mouse common carotid arteries].

OBJECTIVE: To compare the roles of vascular cell adhesion molecular-1 (VCAM-1) and P-selectin in stretch-induced HL-60 cell adhesion to mouse common carotid arteries. METHODS: After the isolated mouse common carotid arteries were perfused with HL-60 cells, the unbound leukocytes were washed away and the number of adherent cells in every single field was counted under a microscope. We first observed the effect of stretch on HL-60 cell adhesion to the endothelium of arteries under different magnitudes of luminal hydrostatic pressure. Then, the roles of VCAM-1 and P-selectin in stretch-induced HL-60 cell adhesion to mouse common carotid arteries were compared by the pretreatment of neutralizing antibodies against VCAM-1, P-selectin or isotype-matched control antibody, respectively. RESULTS: The increase in luminal hydrostatic pressure of carotid arteries led to the increase of the number of HL-60 adhesion cells, suggesting that stretch induced HL-60 cell adhesion in a magnitude-dependent manner. The pretreatment with the isotype-matched control antibody failed to induce a significant change of the number of HL-60 adhesion cells. In contrast, the pretreatment of both VCAM-1 and P-selectin neutralizing antibodies dramatically reduced the number of adherent leukocytes (P<0.05), although no statistical significance was observed between the two groups. CONCLUSION: Stretch can induce HL-60 cell adhesion to carotid arteries in a magnitude-dependent manner. VCAM-1 and P-selectin play some roles in mechanical stretch-induced HL-60 cell adhesion to mouse common carotid arteries.

[An effective method of isolation and primary culture of human umbilical arterial endothelial cells].

OBJECTIVE: To explore and establish an efficient system for isolation and culture of human umbilical arterial endothelial cells (HUAECs). METHODS: The human umbilical arteries were perfused and washed by PBS, and then digested by 0.1 g/L type 1 collagenase. Digested cell supernatants were collected, centrifuged, and the re-suspended cells were cultured in a specific medium. The identification of endothelial cells was carried out by observing the cellular morphology, immunostaining the endothelial surface marker CD31 under fluorescent microscopy and examining the capability to form tube-like structure. RESULTS: The isolated cells grew in a monolayer and exhibited mosaic-like pattern under an inverted phase-contrast microscope. They strongly expressed CD31, an endothelium-specific surface marker, and had an obvious capability to form tube-like structure. CONCLUSION: The method established for isolation of HUAECs in this experiment is simple, rapid and efficient. In addition, the isolated cells are of high purity and high survival.

Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser¹¹77, Ser6³³ and Ser6¹5 and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.

Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways.

Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

[Non-angiogenic functions of vascular endothelial growth factor].

Vascular endothelial growth factor (VEGF or VEGF-A), also named as vascular permeable factor (VPF), is a multi-functional bio-macromolecule belonging to the family of secreted glycoprotein growth factor. VEGF can induce a variety of cellular responses through two high-affinity tyrosine kinases, VEGFR1 and VEGFR2. VEGF plays a key role in the angiogenesis and development in the embryo phase, promoting the proliferation, migration, tube formation and survival of the vascular endothelial cells. In the adult phase, VEGF mainly participates in maintaining the vascular structure and regulating physiological and pathological angiogenesis. Clinical data showed that VEGF signaling inhibitors significantly induced the degeneration of the tumor vessels and reduced tumor size. Meanwhile, various side-effects also have been observed in some patients, indicating that the non-angiogenesis functions of VEGF should be greatly emphasized, especially when developing anti-cancer drugs. Several studies showed that VEGF plays essential roles in various adult organs, such as small intestine, pancreatic islets, thyroid, kidney and liver. When VEGF level in these organs is lower than normal, the complexity of capillary network will be partially degenerated. Apart from that, VEGF also promotes the bone marrow formation, tissue repair and regeneration, the maturation of ovarian, and participates in some pathological courses such as thrombosis, inflammation and ischemia. This review focuses on the non-angiogenesis functions of VEGF and briefly discusses the molecular mechanisms.

VEGF non-angiogenic functions in adult organ homeostasis: therapeutic implications.

Vascular endothelial growth factor (VEGF) is best known as an angiogenic factor essential for embryonic vasculogenesis and postnatal angiogenesis. Considerable evidence has accumulated that VEGF also has non-angiogenic functions. Early studies demonstrated that VEGF transcripts are ubiquitously expressed, and the phosphorylation of VEGF receptor is detectable in adult organs that have no feature of angiogenesis. Recent clinical studies showed that the inhibition of VEGF signaling results in diverse angiogenesis-irrelevant side effects involving the dysfunctions of many organs, suggesting non-angiogenic roles of VEGF in the regulation of organ homeostasis. On the other hand, VEGF stimulates endothelial cells (ECs) to express intercellular adhesion molecules that mediate physical interactions with adjacent tissue cells, or secreted various multifunctional substances that affect the functions of surrounding organs. Furthermore, very recent studies including ours have revealed VEGF as a potent agonist for endothelial exocytosis of Weibel-Palade bodies in which thrombogenic and inflammatory factors are stored. In this brief review, we highlight the importance of VEGF non-angiogenic functions in the modulation of tissue repair and organ regeneration, vascular homeostasis, and inflammation, and propose that the non-angiogenic functions are primarily mediated through the substances released from ECs.

Grb-2-associated binder 1 (Gab1) regulates postnatal ischemic and VEGF-induced angiogenesis through the protein kinase A-endothelial NOS pathway.

The intracellular signaling mechanisms underlying postnatal angiogenesis are incompletely understood. Herein we show that Grb-2-associated binder 1 (Gab1) plays a critical role in ischemic and VEGF-induced angiogenesis. Endothelium-specific Gab1 KO (EGKO) mice displayed impaired angiogenesis in the ischemic hindlimb despite normal induction of VEGF expression. Matrigel plugs with VEGF implanted in EGKO mice induced fewer capillaries than those in control mice. The vessels and endothelial cells (ECs) derived from EGKO mice were defective in vascular sprouting and tube formation induced by VEGF. Biochemical analyses revealed a substantial reduction of endothelial NOS (eNOS) activation in Gab1-deficient vessels and ECs following VEGF stimulation. Interestingly, the phosphorylation of Akt, an enzyme known to promote VEGF-induced eNOS activation, was increased in Gab1-deficient vessels and ECs whereas protein kinase A (PKA) activity was significantly decreased. Introduction of an active form of PKA rescued VEGF-induced eNOS activation and tube formation in EGKO ECs. Reexpression of WT or mutant Gab1 molecules in EGKO ECs revealed requirement of Gab1/Shp2 association for the activation of PKA and eNOS. Taken together, these results identify Gab1 as a critical upstream signaling component in VEGF-induced eNOS activation and tube formation, which is dependent on PKA. Of note, this pathway is conserved in primary human ECs for VEGF-induced eNOS activation and tube formation, suggesting considerable potential in treatment of human ischemic diseases.