Endothelial Cell-conditioned Medium Research Articles

The Lymphatic Endothelial Cell Secretome Inhibits Osteoblast Differentiation and Bone Formation.

Complex lymphatic anomalies (CLAs) are a set of rare diseases with unique osteopathic profiles. Recent efforts have identified how lymphatic-specific somatic activating mutations can induce abnormal lymphatic formations that are capable of invading bone and inducing bone resorption. The abnormal bone resorption in CLA patients has been linked to overactive osteoclasts in areas with lymphatic invasions. Despite these findings, the mechanism associated with progressive bone loss in CLAs remains to be elucidated. In order to determine the role of osteoblasts in CLAs, we sought to assess osteoblast differentiation and bone formation when exposed to the lymphatic endothelial cell secretome. When treated with lymphatic endothelial cell conditioned medium (L-CM), osteoblasts exhibited a significant decrease in proliferation, differentiation, and function. Additionally, L-CM treatment also inhibited bone formation through a neonatal calvaria explant culture. These findings are the first to reveal how osteoblasts may be actively suppressed during bone lymphatic invasion in CLAs.

Abstract 3177: Liver endothelium secreted LRG1 is a novel ligand of HER3 to promote metastatic colorectal cancer growth

Abstract Background: Liver is the most common site of developing distant metastasis in colorectal cancer (CRC). Patients with metastatic CRC (mCRC) have a 5-year survival rate at 14%. We previously showed that liver endothelial cells (EC), a key component of the liver microenvironment, secrete soluble factors to promote CRC growth and chemoresistance via activating human epidermal growth factor receptor (ERBB3, also known as HER3). However, we found that the ECs activated HER3 by a previously unknown mechanism that is independent of the established HER3 ligand neuregulins. Identifying a new HER3 ligand will potentially give rise to novel therapeutic target for treating patients with mCRC. Methods: We first fractionated conditioned medium (CM) from liver ECs by fast protein liquid chromatography. The specific fractions that activated HER3 and AKT in CRC cells were subjected to mass spectrometry (MS) for protein identification. In parallel, we used a His-tagged HER3 Extracellular domain (ECD) and pulled down EC-secreted factors that directly bound to HER3 and then subjected to protein identification by MS. Candidate proteins identified in both MS analyses were subjected for further validations by siRNA or immunoprecipitation depletion from EC CM to determine their roles in HER3 activation and CRC growth. Results: We identified a specific fraction of liver EC CM increased phosphorylation of HER3 and AKT in CRC cells (determined by Western blotting). Meanwhile, the HER3-ECD affinity binding assay showed that HER3 ECD-depleted EC CM could no longer activate HER3 or promote growth, and the soluble factor(s) eluted from HER3-ECD increased phosphorylation of HER3 and AKT, and promoted growth in CRC cells. Results from both MS analyses revealed that LRG1 is a potential ligand that binds to and activate HER3 in CRC cells. We depleted LRG1 from EC CM, either by siRNA knockdown LRG1 in ECs or immunoprecipitation, and found that LRG1-depleted EC CM could no longer activate HER3 and AKT in CRC cells, and LRG1-depleted EC CM failed to promote growth of CRC cell in vitro and CRC xenografts in vivo. Conclusions: We identified LRG1 as a new HER3 ligand and demonstrated that liver EC-secreted LRG1 mediates EC paracrine effects on activating HER3 and promoting CRC cell growth. Our findings suggest an oncogenic role of EC-secreted LRG1 in the surrounding liver microenvironment of mCRC, and highlighted a potential of blocking LRG1 with monoclonal antibodies for treating patients with mCRC. Citation Format: Moeez Ghani Rathore, Wei Zhang, Michel'le Wright, Jordan Winter, Yamu Li, Zhenghe Wang, Rui Wang. Liver endothelium secreted LRG1 is a novel ligand of HER3 to promote metastatic colorectal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3177.

Abstract 2552: Effects of liver endothelium on pancreatic cancer growth and metabolism

Abstract Introduction: ~90% metastatic pancreatic ductal adenocarcinoma (mPDAC) are found in the liver, and 5-year survival rate for patients with mPDAC is only at 3%. Therefore, novel therapeutic strategies are urgently needed. A growing body of evidence suggest PDAC rely on mitochondrial function (oxidative phosphorylation, OXPHOS) for survival. However, PDAC liver metastases have been reported to have higher levels of glucose uptake compared to primary tumors suggesting that PDAC liver metastases may rely on glycolytic metabolism. Therefore, the relative metabolic and cellular profiles of primary and metastatic PDAC (mPDAC) remain unclear and the involved regulatory pathway(s) have not been elucidated. Our previous studies showed that liver endothelial cells (ECs) secreted soluble factors to promote the survival of colon cancer cells in a paracrine fashion. The influence of the liver EC microenvironment on mPDAC growth and metabolism has not been elucidated. In this study, we investigate the paracrine effects of liver ECs on the survival and metabolic profiles of PDAC and elucidate the involved mechanism(s). Methods: Primary liver ECs were isolated from non-neoplastic liver. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Effects of EC CM on PDAC metabolism was assessed by ATP production with CellTiter-Glo and oxygen consumption rate with Agilent Seahorse Mito Stress Assay. Results: Compared to PDAC CM, liver EC CM promoted proliferation in different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3, also known asERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, significantly blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. Furthermore, EC CM decreased the levels of ATP production and O2 consumption in PDAC cells, suggesting that EC cells reprogram PDAC metabolism away from OXPHOS mitochondrial metabolism. Conclusions: Liver EC-secreted factors promoted PDAC growth in vitro and in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EC reprogramed PDAC metabolism towards glycolysis, and inhibiting the activation of HER3 shifted PDAC metabolism to OXPHOS. Our findings provide a new insight to develop new combination of HER antibody and OXPHOS inhibitor for treating patients with HER3+ve mPDAC. Citation Format: Wei Zhang, Michel'le Wright, Moeez Rathore, Mehrdad Zarei, Jordan Winter, Rui Wang. Effects of liver endothelium on pancreatic cancer growth and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2552.

Extracellular Hsp90α, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes

IntroductionAtherosclerosis is the main pathological change in diabetic angiopathy, and vascular inflammation plays an important role in early atherosclerosis. Extracellular heat shock protein 90 (eHsp90) is secreted into the serum...

Abstract PO-131: The role of liver endothelium on pancreatic cancer growth

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among major cancers in the United States, and the 5-year survival rate for patients with metastatic PDAC (mPDAC) is only at 3%. Past studies have shown that the paracrine secretion of soluble factors by endothelial cells (ECs) created a unique niche and promoted the survival of cancer cells (cell growth or chemoresistance) in other types of cancer. The liver is the main site of distant metastases in mPDAC, but the influence of the liver EC microenvironment on mPDAC has not been elucidated. In this study, we determined the paracrine effects of liver ECs on the survival of PDAC and identify involved mechanism. Methods: Primary liver ECs were isolated from non-neoplastic liver tissues to mimic the liver EC microenvironment. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Involved RKTs were blocked by antibodies for determining their roles in mediating EC effects on PDAC cells. Lastly, A xenograft tumor model was used to establish PDAC tumors and then treated xenograft mice were subcutaneously injected by either PADC CM or EC CM, and tumors growth was monitored and recorded to evaluate the effect of ECs on PADCs. Results: Compared to PDAC CM, EC CM promoted proliferation in 4 different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3 or ERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, completely blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. It is interesting that ERK activation was also observed but was not affected by HER3 inhibition. It implied that EGFR signaling pathway might also be involved in EC CM induced PDAC cell growth. Moreover, the combination of cetuximab, trastuzumab and seribantumab yielded the best inhibitory ability on EC CM promoted cell growth as compared to antibody alone or the combination of two of them. Finally, EC CM promoted PDAC tumor growth was also observed in BxPC-3 derived xenograft mouse model. Conclusions: Our results demonstrated that liver EC-secreted factors promoted PDAC growth either in vitro or in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EGFR pathway may also play a role in EC induced cell growth and needs to be addressed in the future study. Our findings suggest a potential of using the combination of HER antibodies/inhibitors for treating patients with HER3+ve mPDACs. Citation Format: Wei Zhang, Michel’le Wright, Moeez Rathore, Ali Vaziri-Gohar, Jordan Winter, Rui Wang. The role of liver endothelium on pancreatic cancer growth [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-131.

Autocrine and Paracrine Effects of Vascular Endothelial Cells Promote Cutaneous Wound Healing.

Background When vascular endothelial cells are subjected to external stimuli, paracrine hormones and cytokines act on adjacent cells. The regulation of the biological behaviour of cells is closely related to the maintenance of organ function and the occurrence and development of disease. However, it is unclear whether vascular endothelial cells affect the biological behaviour of cells involved in wound repair through autocrine and paracrine mechanisms and ultimately play a role in wound healing. We aimed to verify the effect of the autocrine and paracrine functions of vascular endothelial cells on wound healing. Materials and Methods ELISA was used to detect platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor in human umbilical vascular endothelial cell-conditioned medium (HUVEC-CM). Different concentrations of HUVEC-CM were used to treat different stem cells. CCK-8 and scratch assays were used to detect the proliferation and migration ability of each cell. A full-thickness dorsal skin defect model was established in mice, and skin wound healing was observed after the local injection of HUVEC-CM, endothelial cell medium (ECM), or normal saline. H&E staining and immunofluorescence were used to observe the gross morphology of the wound tissue, the epithelial cell migration distance, and the expression of CD3 and CD31. Results HUVEC-CM promotes the proliferation and migration of epidermal stem cells, skin fibroblasts, bone marrow mesenchymal stem cells, and HUVECs themselves. Furthermore, HUVEC-CM can promote angiogenesis in mouse skin wounds and granulation tissue formation and can accelerate wound surface epithelialization and collagen synthesis, thereby promoting wound healing. Conclusion Our results clearly suggest that it is practicable and effective to promote wound healing with cytokines secreted by vascular endothelial cells in a mouse model.

Endothelial cell secreted VEGF-C enhances NSC VEGFR3 expression and promotes NSC survival

Although delivery of neural stem cell (NSC) as a therapeutic treatment for intracerebral hemorrhage (ICH) provides promise, NSC delivery typically has extremely low survival rates. Here, we investigate endothelial cell (EC) and pericyte (PC) interactions with NSC, where our results demonstrate that EC, and not PC, promote NSC cell proliferation and reduce cytotoxicity under glucose deprivation (GD). Additionally, NSC proliferation was increased upon treatment with EC conditioned media, inhibited with antagonism of VEGFR3. In an NSC + EC co-culture we detected elevated levels of VEGF-C, not seen for NSC cultured alone. Exogenous VEGF-C induced NSC upregulation of VEGFR3, promoted proliferation, and reduced cytotoxicity. Finally, we delivered microbeads containing NSC + EC into a murine ICH cavity, where VEGF-C was increasingly present in the injury site, not seen upon delivery NSC encapsulated alone. These studies demonstrate that EC-secreted VEGF-C may promote NSC survival during injury, enhancing the potential for cell delivery therapies for stroke.

Flow-dependent differentiation of cultured adrenal cells under different stimuli.

It still remains unclear how the functional organisation of the adrenal cortex arises. One aim of this study was to create a setup which allows for the establishment of a concentration gradient in vitro. This was achieved by a continuous flow of medium through the culture flask which caused differences in glucose and cortisol concentrations as well as in pH values between the sites of inflow and outflow of medium. Using real-time polymerase chain reaction, we found that a continuous supply of 1ml medium per hour significantly increased the expression of MC2R, CYP11B1 and CYP17A1 genes of NCI-H295R cells in the distal area of the flask as compared with the proximal part. The expression of the AT1R showed a reverse regulation. The addition of dexamethasone to the medium led to an increase in gene expression of MC2R while AT1R was downregulated. Moreover, we detected a higher expression of CYP11B2 and a decreased expression of CYP11B1 when endothelial cell-conditioned medium (ECCM) was added to the inflow. Our experiments show that a directed medium delivery system creates different gradients and affects the functional differentiation of the NCI-H295R cells. Also, our results emphasise that products of endothelial cells have additional effects on the differentiation of the cultured adrenal cortical cells. Our results are in support that the regulation of the adrenal zonation is possible through different concentration gradients.

Abstract PS16-15: The influence of mechanical and chemical insult on the integrity of vascular endothelial cells and associated impact on epithelial protein lost in neoplasm (EPLIN) and potential partner protein network

Abstract Background. Vascular endothelium is the frontline structure in the body facing constant high velocity flow and external harmful agents absorbed within the circulation. It is not fully understood how endothelial cells respond to such external insults. We have previously shown that a cytoskeleton binding protein, Epithelial Protein Lost in Neoplasm (EPLIN) is an essential protein in regulating the migration and adhesiveness of vascular endothelial cells and in doing so, contributing to the normal process of angiogenesis and its responsiveness to complex environments, thereby influencing tumour development (1). The portraited role of EPLIN in the intracellular process has led to the hypothesis that EPLIN has an important role to play during the response of endothelial cells to external hostile factors by providing a stable and coordinated mechanism. The current study explores the influence of EPLIN and its associated protein network in the endothelial cell response to physical injury and extrinsic carcinogenic agents. Method. Human primary cultured vascular endothelial cells and immortalised endothelial cells were used as cell models. The cells were subject to either physical wounding injuries or exposure to a carcinogenic chemical, namely diethystibestrol (DIET). Gene expression patterns from the endothelial cells were subject to analysis by gene microarray. The cellular response of endothelial cells was explored using proliferation and migration assays. The potential paracrine impact on cancer cells in close proximity to the endothelial cells was tested using endothelial cell conditioned media. Results. Both mechanical injury and chemical challenge (at non-toxic concentration range) resulted in mild yet statistically insignificant reductions in EPLIN expression in both endothelial models. A number of the proposed EPLIN network partners in immortalised endothelial cells were affected by mechanic injuries, most notably CTNNA1, CTNNAL1, CTNNA3, CTNNB1 and the caveolins. Chemical injury resulted in more drastic reductions of caveolin and its variant2 (CAV2) (p<0.001 for both). In the cell model, neither mechanical nor chemical insults caused significant changes in the regulatory elements of the network, namely signal-induced proliferation-associated gene 1 (SIPA1) nor its variants. Likewise, other network members closely linked to the cytoskeleton, including gamma-catenin and paxillin, were also unaffected. A similar response was seen with primary culture endothelial cells. Endothelial cells, following mechanical injury had a stimulatory effect on the growth of breast cancer cells (MDA MB-231 and BT20) due largely to a rapid rise in G2 and S phase of cell cycle and migration of breast cancer cells, in a co-culture assay. However, the effects of endothelial cells treated with DIET elicited more profound effects on the migration of cancer cells. Discussion. Our results demonstrate that vascular endothelial cells are sensitive to the external mechanical and harmful chemical damages. Such damage appears to influence breast cancer cells in a paracrine fashion. Similarly, such damage resulted in altered expression of a range of cell adhesion molecules but did not significantly influence EPLIN. EPLIN, a central element of this network may be involved in co-ordinating this response though further study is needed. Reference1 Sanders AJ, Ye L, Mason MD, et al. The impact of EPLINα (Epithelial protein lost in neoplasm) on endothelial cells, angiogenesis and tumorigenesis. Angiogenesis. 2010 Dec;13(4):317-26. Citation Format: Jianyuan Zeng, Andrew James Sanders, Tracey Amanda Martin, Wen Guo Jiang. The influence of mechanical and chemical insult on the integrity of vascular endothelial cells and associated impact on epithelial protein lost in neoplasm (EPLIN) and potential partner protein network [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-15.

Tomoh Masaki

Tomoh Masaki

Endothelial cell modulation of cardiomyocyte gene expression

The anatomic arrangement of microvascular endothelial cells and cardiomyocytes in vivo enables close interactions among these cells. In our in vitro co-culture system, ANP and BNP expression in the mouse atrial cardiomyocyte cell line HL-1 and subsequent ANP release were significantly upregulated when co-cultured with mouse cardiac microvascular endothelial cells or exposed to endothelial cell-conditioned medium. Endothelin-1 (ET-1) activation of endothelial cells remarkably enhanced their paracrine effect on cardiomyocyte gene expression, suggesting that ET-1 stimulation of endothelial cells affects expression of fetal genes such as ANP and BNP in adult cardiomyocytes through paracrine signalling. Exposure of HL-1 cells and murine induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to authentic angiopoietin-2 (Ang2) caused a concentration-dependent decrease in ANP expression while ET-1-induced ANP expression was augmented by low but inhibited by high concentrations of Ang2. FK506-mediated inhibition of the calcineurin-NFAT pathway in the HL-1 cells selectively inhibited the stimulatory effect of the conditioned medium derived from ET-1-pre-stimulated endothelial cells on cardiomyocyte fetal gene expression. Combined with previous results indicating a crucial role for ANP and BNP in cardiac homeostasis, our findings provide further evidence that paracrine signalling by cardiac microvascular endothelial cells modulates cardiomyocyte function.

Abstract 5163: Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status

Abstract INTRODUCTION: Colorectal cancer (CRC) is the second-leading cause of cancer-related death in the United States. A better understanding of the regulation of CRC cell survival pathways is necessary for developing new therapeutic strategies for patients with metastatic CRC (mCRC). We have previously found that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in their conditioned medium (CM) that, in turn, increase cell growth and chemoresistance of CRC cells. We also determined that EC-induced CRC cell survival is mediated, in part, by HER3/ERRB3 signaling. OBJECTIVES: In the current study we sought to determine if alterations in driver mutations mediate the role of liver ECs in activating HER3 signaling and promoting cell survival in CRC cells. METHODS: Primary ECs from non-malignant liver parenchyma were isolated as previously described (Wang et al., Mol Cancer Res., 2018). CRC cell lines with distinct molecular profiles (MSI status, and wildtype or mutant KRAS, BRAF, and PIK3CA) were incubated with either their own CM (control) or CM from ECs. The effects of CM on CRC cell growth and response to 5-fluorouracil (5-FU) was determined by MTT and colony formation assays. The effect of angiocrine signaling on chemoresistance was also determined by fluorescence-activated cell sorting (FACS) for apoptosis after treating CRC cells with 5-FU, either in control CM or EC CM. HER3 blockade was accomplished by 1) siRNA knockdown or 2) a HER3 antibody. RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells regardless of the gene alteration profiles in in vitro assays. We confirmed that EC CM activated the HER3-AKT signaling pathway in CRC cells. Furthermore, we found that HER3 blockade attenuated EC CM-induced AKT activation and cell survival in CRC cells independent of the cell mutational profile. CONCLUSIONS: Our results demonstrate that HER3 mediates liver EC-induced cell growth and chemoresistance in CRC cells independent of the driver mutational profiles of the cells. The genetic background of CRC cells is not a mediator of EC CM-induced HER3 activation and cell survival. These findings suggest that effects of HER3 targeting of tumor cells in patients with mCRC should be independent of MSI status, or RAS, BRAF, or PIK3CA mutational status. Citation Format: Rui Wang, Rajat Bhattacharya, Fan Fan, Xiangcang Ye, Lee M. Ellis. Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5163.

MicroRNA-145 Regulates the Differentiation of Adipose Stem Cells Toward Microvascular Endothelial Cells and Promotes Angiogenesis

Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in ischemic tissues. However, the mechanism that promotes ASCs differentiation toward endothelial cells (ECs) is not known. To investigate the mechanisms of ASCs differentiation into ECs. ASCs were isolated from clinical lipoaspirates and cultured with DMEM or endothelial cell-conditioned medium. Endothelial cell-conditioned medium induced downregulation of miR-145 in ASCs and promoted endothelial differentiation. We identified bFGF (basic fibroblast growth factor) released by ECs as inducer of ASCs differentiation through receptor-induced AKT (protein kinase B) signaling and phosphorylation of FOXO1 (forkhead box protein O1) suppressing its transcriptional activity and decreasing miR-145 expression. Blocking bFGF-receptor or PI3K/AKT signaling in ASCs increased miR-145 levels. Modulation of miR-145 in ASCs, using a miR-145 inhibitor, regulated their differentiation into ECs: increasing proliferation, migration, inducing expression of EC markers (VE-cadherin, VEGFR2 [vascular endothelial growth factor receptor 2], or VWF [von Willebrand Factor]), and tube-like formation. Furthermore, in vivo, downregulation of miR-145 in ASCs enhanced angiogenesis in subcutaneously implanted plugs in mice. In a murine hindlimb ischemia model injection of ASCs with downregulated miR-145 induced collateral flow and capillary formation evidenced by magnetic resonance angiography. Next, we identified ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) as the target of miR-145. Upregulation of miR-145 in ASCs, by mimic miR-145, suppressed ETS1 expression and consequently abolished EC differentiation and the angiogenic properties of endothelial cell-conditioned medium-preconditioned ASCs; whereas, overexpression of ETS1 reversed the abrogated antiangiogenic capacity of miR-145. ETS1 overexpression induced similar results to those obtained with miR-145 knockdown. bFGF released by ECs induces ASCs differentiation toward ECs through miR-145-regulated expression of ETS1. Downregulation of miR-145 in ASCs induce vascular network formation in ischemic muscle.

The associations of endothelial and podocyte injury in proliferative lupus nephritis: from observational analysis to in vitro study.

Our study aims to evaluate the endothelial cell-podocyte crosstalk in proliferative lupus nephritis (LN). The semi-quantification scores of glomerular endothelial cell injury and the foot process width (FPW) were processed in 110 proliferative LN patients. Podocytes were stimulated with LN-derived IgG. Glomerular endothelial cells were treated with podocyte-conditioned medium (PCM), and then podocytes were incubated with endothelial cell-conditioned medium (ECM). The levels of vascular endothelial growth factor-A (VEGF-A) in PCM and endothelin-1 in ECM were analyzed, and the injury of podocyte and glomerular endothelial cells were further evaluated. The pathological score of glomerular endothelial cell injury was correlated with FPW in LN complicated with thrombotic microangiopathy. In vitro study showed the following: 1. Stimulation of podocytes by IgG from LN led to decline in the expression of nephrin with cytoskeleton rearrangement, and reduction of VEGF-A levels. 2. Exposure of glomerular endothelial cells to PCM incubated with LN-derived IgG (PCM-LN) induced more endothelin-1 secretion and disruption of intercellular tight junction. 3. Exposure of podocytes to ECM stimulated with PCM-LN could induce cytoskeleton redistribution with decrease of nephrin. In conclusion, the pathological glomerular endothelial cell lesions were associated with FPW and the VEGF-endothelin-1 system might play a critical role in the endothelial cell-podocyte crosstalk in LN.

Endothelial Cells Promote Colorectal Cancer Cell Survival by Activating the HER3-AKT Pathway in a Paracrine Fashion.

The regulation of colorectal cancer cell survival pathways remains to be elucidated. Previously, it was demonstrated that endothelial cells (EC) from the liver (liver parenchymal ECs or LPEC), the most common site of colorectal cancer metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in colorectal cancer cells. However, the paracrine effects of LPECs on other colorectal cancer cellular functions have not been investigated. Here, results showed that CM from LPECs increased cell growth and chemoresistance by activating AKT in colorectal cancer cells in vitro. Using an unbiased receptor tyrosine kinase array, it was determined that human epidermal growth factor receptor 3 (ERBB3/HER3) was activated by CM from LPECs, and it mediated AKT activation, cell growth, and chemoresistance in colorectal cancer cells. Inhibition of HER3, either by an inhibitor AZD8931 or an antibody MM-121, blocked LPEC-induced HER3-AKT activation and cell survival in colorectal cancer cells. In addition, CM from LPECs increased in vivo tumor growth in a xenograft mouse model. Furthermore, inhibiting HER3 with AZD8931 significantly blocked tumor growth induced by EC CM. These results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in colorectal cancer cells. IMPLICATIONS: This study suggested a potential of treating patients with metastatic colorectal cancer with HER3 antibodies/inhibitors that are currently being assessed in clinical trials for various cancer types.

Endothelial cells exposed to phosphate and indoxyl sulphate promote vascular calcification through interleukin-8 secretion.

Vascular calcification (VC) is amplified during chronic kidney disease, partly due to uraemic toxins such as inorganic phosphate (Pi) and indoxyl sulphate (IS) that trigger osteogenic differentiation of vascular smooth muscle cells (VSMCs). These toxins also alter endothelial cell (EC) functions but whether this contributes to VC is unknown. Here, we hypothesized that ECs exposed to Pi and IS promote VSMC calcification. Human umbilical vein ECs were treated with Pi, IS or both, and then the conditioned media [endothelial cell conditioned medium (EC-CM)] was collected. Human aortic SMCs (HASMCs) were exposed to the same toxins, with or without EC-CM, and then calcification and osteogenic differentiation were evaluated. Procalcifying factors secreted from ECs in response to Pi and IS were screened. Rat aortic rings were isolated to assess Pi+IS-induced calcification at the tissue level. Pi and Pi+IS induced HASMCs calcification, which was significantly exacerbated by EC-CM. Pi+IS induced the expression and secretion of interleukin-8 (IL-8) from ECs. While IL-8 treatment of HASMCs stimulated the Pi+IS-induced calcification in a concentration-dependent manner, IL-8 neutralizing antibody, IL-8 receptors antagonist or silencing IL-8 gene expression in ECs before collecting EC-CM significantly prevented the EC-CM procalcifying effect. IL-8 did not promote the Pi+IS-induced osteogenic differentiation of HASMCs but prevented the induction of osteopontin (OPN), a potent calcification inhibitor. In rat aortic rings, IS also promoted Pi-induced calcification and stimulated the expression of IL-8 homologues. Interestingly, in the Pi+IS condition, IL-8 receptor antagonist lifted the inhibition of OPN expression and partially prevented aortic calcification. These results highlight a novel role of IL-8, whose contribution to VC in the uraemic state results at least from interaction between ECs and VSMCs.

Abstract 191: Paracrine role of endothelial cells in HER3-mediated colon cancer cell survival

Abstract INTRODUCTION: Colorectal cancer (CRC) remains the second-leading cause of cancer-related death in the United States. Median survival of patients with metastatic CRC (mCRC) is ~2.5 years and patients often become resistance to systemic therapy within 1 year of treatment. Therefore, a better understanding of the regulation of CRC cell survival pathways is necessary in the development of new therapeutic strategies that will improve outcomes for patients with mCRC. We have previously demonstrated that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in CRC cells. OBJECTIVES: In this study, we sought to (1) elucidate the paracrine role of liver ECs in mediating CRC cell growth and chemoresistance, and (2) determine the mechanism(s) involved. METHODS: Primary ECs from non-malignant liver parenchyma were isolated and confirmed. CRC cells were incubated with either their own CM (control) or CM from ECs. The effect of CM on CRC cell growth was determined by the MTT assay. The effect on chemoresistance was determined by fluorescence-activated cell sorting (FACS)-based assay for apoptosis after treating CRC cells with 5-fluorouracil (5-FU) either in control CM or EC CM. Effects of CM on CRC tumor growth in vivo was determined using a subcutaneous (subQ) xenograft tumor model. CRC cells were pretreated with control or EC CM and then injected subQ in an inoculation mixture of Matrigel and concentrated CM. Tumor burden was assessed over time by bioluminescence with in vitro imaging system (IVIS) and by measuring tumor volume with calipers. RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells in vitro via activating the AKT pathway. We identified human epidermal growth factor receptor 3 (HER3, also known as ERBB3) being the only receptor tyrosine kinase (RTK) activated by EC CM. Furthermore, we found that inhibition of HER3, either by a HER3 inhibitor or by HER3 siRNAs, blocked EC CM-induced AKT activation, cell growth, and chemoresistance in CRC cells. In our in vivo tumor model, CRC cells treated with EC CM formed larger tumors with higher growth rates than that with control CM. CONCLUSIONS: Our results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in CRC cells. Identification of the liver EC-secreted factor(s) for activating CRC-associated HER3 is currently under study. This study suggested a potential strategy of treating mCRC patients with HER3 antibodies/inhibitors that are already being assessed in clinical trials for various cancer types. Citation Format: Rui Wang, Rajat Bhattacharya, Fan Fan, Xiangcang Ye, Delphine Boulbes, Lee M. Ellis. Paracrine role of endothelial cells in HER3-mediated colon cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 191.

Mesenchymal stem cells microvesicles stabilize endothelial barrier function partly mediated by hepatocyte growth factor (HGF)

BackgroundMesenchymal stem cells microvesicles (MSC-MVs) stabilize endothelial barrier function in acute lung injury (ALI); however, the detailed mechanism remains to be further defined. Hepatocyte growth factor (HGF), which is derived from MSC-MVs, might have a key role in the restoration of endothelial barrier function by MSC-MVs.MethodsMSCs with lentiviral vector-mediated HGF gene knockdown (siHGF-MSC) were generated. A co-culture model of pulmonary microvascular endothelial cells and MSC-MVs collected from MSCs or siHGF-MSCs after 24 h of hypoxic culture was utilized. Then, endothelial paracellular and transcellular permeabilities were detected. VE-cadherin, and occludin protein expression in the endothelial cells was measured using Western blot. Endothelial cell proliferation was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Endothelial cell apoptosis was analysed using TUNEL assay. Finally, IL-6 and IL-10 production was determined via an enzyme-linked immunosorbent assay (ELISA).ResultsTreatment with MSC-MVs significantly decreased LPS-induced endothelial paracellular and transcellular permeabilities, and the effect was significantly inhibited after HGF gene knockdown in MSC-MVs. Furthermore, treatment with MSC-MVs increased the expression of the endothelial intercellular junction proteins VE-cadherin and occludin. Treatment with MSC-MVs also decreased endothelial apoptosis and induced endothelial cell proliferation. Finally, the treatment reduced IL-6 production and increased IL-10 production in the conditioned media of endothelial cells. However, the effects of the treatment with MSC-MVs were inhibited after HGF gene knockdown.ConclusionsMSC-MVs protect the barrier functions of pulmonary microvascular endothelial cells, which can be partly attributed to the presence of HGF in the MSC-MVs.

Abstract 1806: Endothelial cells activate the cancer stem cell-associated Nanogp8 pathway in colorectal cancer cells in a paracrine fashion

Abstract INTRODUCTION: Median survival of patients with metastatic colorectal cancer (mCRC) is ~2.5 years and patients often become resistance to systemic therapy within 1 year of the diagnosis of metastasis. There is accumulating evidence for the existence of cancer stem cells (CSCs) in CRC, which are now believed to mediate chemoresistance. The molecular mechanisms that regulate the CSC phenotype in CRC have not been fully elucidated. We have previously described that endothelial cells (ECs) from liver, the most common site of metastases of CRC, secreted soluble Jagged-1 peptides to activate Notch signaling and increase the CSC phenotype of CRC cells in a paracrine fashion. In the current project, we sought to further elucidate the paracrine roles of liver ECs and ECs from other organs in mediating specific CSC-associated pathways in CRC cells. METHODS: Several EC lines from liver, lung, colon mucosa and kidney were established and used to determine if conditioned medium (CM) of ECs from distinct organs promotes the CSC phenotype and chemoresistance in CRC cells. CRC cells were incubated with either their own CM (control) or CM from ECs. The CSC phenotype was determined by sphere formation assays. Chemoresistance was determined by Western blotting for apoptotic markers and fluorescence-activated cell sorting (FACS)-based Annexin V-PI double staining assay for apoptosis after treating CRC cells with 5-fluorouracil (5-FU) either in control CM or CM of ECs. The effects of EC CM on CSC-associated genes were determined by unbiased qPCR array and validated by Western blotting. RESULTS: CM from ECs from liver and other organs all significantly increased sphere formation in CRC cells. qPCR arrays and Western blotting showed that the CSC-associated Nanog and Oct4 genes were increased by EC CM. Moreover, CRC cells treated by liver EC CM became more resistant to 5-FU induced apoptosis. Luciferase reporter assays suggested that the Nanog proteins in CRC cells were encoded by the retrogene NANOGP8, and EC CM specifically induced the expression of NANOGP8 but not NANOG. The specific induction of NANOGP8 in CRC cells were confirmed by AlwNI digestion, an established technique to distinguish NANOG and NANOGP8 based on a single nucleotide polymorphism (SNP) between two genes. In addition, Akt was shown to mediate the induction of NANOGP8 in CRC cell, as the PI3K inhibitor wortmannin blocked liver EC CM-induced NanogP8 expression and blocked sphere formation in CRC cells. CONCLUSION: Our studies demonstrated a paracrine role of ECs from several organs, including liver, in regulating the CSC phenotype in CRC cells, via increasing NanogP8 expression. This study elucidated the importance of ECs in the microenvironment in mediating the survival of metastatic CRC cells in the liver, and potentially other organs. Citation Format: Rui Wang, Rajat Bhattacharya, Xiangcang Ye, Delphine R. Boulbes, Fan Fan, Lee M. Ellis. Endothelial cells activate the cancer stem cell-associated Nanogp8 pathway in colorectal cancer cells in a paracrine fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2017-1806

Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes.

Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.