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Background: The early noninvasive screening of patients suitable for neoadjuvant chemotherapy (NCT) is essential for personalized treatment in locally advanced gastric cancer (LAGC). The aim of this study was to develop and visualized a radio-clinical biomarker from pretreatment oversampled CT images to predict the response and prognosis to NCT in LAGC patients.Methods: 1060 LAGC patients were retrospectively recruited from six hospitals from January 2008 to December 2021. The training (TC) and internal validation cohort (IVC) were randomly selected from center I. The external validation cohort (EVC) comprised 265 patients from 5 other centers. An SE-ResNet50-based chemotherapy response predicting system (DL signature) was developed from pretreatment CT images preprocessed with imaging oversampling method (i.e. DeepSMOTE). Then, DL signature and clinic-based features were fed into the deep learning radio-clinical signature (DLCS). The predictive performance was evaluated according to discrimination, calibration and clinical usefulness. Model for OS prediction were built to further explore the survival benefit of the proposed DL signatures and clinicopathological characteristic. Result: DLCS showed perfect performance in predicting the response to NCT in the IVC (AUC, 0.86) and EVC (AUC, 0.82), with good calibration in all cohorts (p > 0.05). In addition, the performance of DLCS was better than that of the clinical model (p＜0.05). Finally, we found that the DL signature could also serve as an independent factor for prognosis (HR, 0.828, p = 0.004). The C-index, iAUC, IBS for the OS model were 0.64, 1.24 and 0.71 in the test set.Conclusion: We proposed the DLCS that links the imaging features to clinical risk factors to generate high accuracy classification of tumor response and risk identification of OS in LAGC patients prior to NCT that then can be used for guiding personalized treatment plans with the help of the visualization of computerized tumor-level characterization.

Tumor angiogenesis is a key step in the progression of gastric cancer (GC) that delivers essential nutrients and oxygen to tumor cells and distant sites. The cyclic AMP responsive element-binding protein 3-like 4 (CREB3L4) is a transcription factor highly expressed in multiple human cancers. This study aimed to investigate the regulatory effects of CREB3L4 on GC progression and angiogenesis. CREB3L4 was overexpressed in GC tissues and cell lines, and was positively correlated with advanced tumor stage and poor survival in GC patients. The upregulation of CREB3L4 in GC cells increased cell viability, promoted cell proliferation, reduced apoptosis, enhanced cell migration and invasion, and induced the formation of tubule-like endothelial structures, whereas CREB3L4 knockdown impeded tumor cell growth, attenuated cell motility, and prevented human umbilical vein endothelial cells from forming tubule-like structures. In addition, mice inoculated with CREB3L4-deficient GC cells showed significantly suppressed tumor growth compared to the group harboring wild-type tumors. Further analysis revealed that CREB3L4 expression was positively correlated with the level of vascular endothelial growth factor A (VEGFA) in gastric tumors. CREB3L4 regulated the transcription activity of VEGFA by binding to its promoter. The downregulation of VEGFA eliminated CREB3L4-induced GC cell growth and movement, and the formation of endothelial structures; while VEGFA upregulation greatly induced the growth and movement of GC cells with CREB3L4 deficiency. In conclusion, CREB3L4 promoted gastric tumor progression and endothelial angiogenesis by transcriptionally activating the VEGFA promoter, suggesting that therapeutic potential of the CREB3L4/VEGFA axis in GC treatment.

Objective To study the effect of lncRNA HAND2-AS1 on gastric adenocarcinoma (GA) cell property and explore its specific mechanism. Methods Data on stomach adenocarcinoma (STAD) were analyzed to screen differentially expressed lncRNA HAND2-AS1. RNA22-HAS database and dual luciferase reporter assay were applied to confirm the target relationship between HAND2-AS1/HIF3A and miR-184. The HAND2-AS1 and miR-184 expressions in tissue or cells were determined by qRT-PCR and Western blot. Besides, after GA cells (AGS) cultured in normoxic and hypoxic condition, phosphoenolpyruvate (PEP) and lactic acid were quantified by Phosphoenolpyruvate Fluorometric Assay Kit and Lactic Acid Detection kit, respectively. Additionally, colony formation assay, transwell invasion and migration assays were used to evaluate the abilities of cell invasion, migration, and proliferation in distinct conditions. Results The HAND2-AS1 and HIF3A expressions were down-regulated and miR-184 expression was up-regulated in GA tissues and cells. Dual luciferase reporter assay confirmed HAND2-AS1 and HIF3A were targeted by miR-184. AGS cell proliferation abilities were restrained by HAND2-AS1 and HIF3A overexpression and enhanced by miR-184, as well as migration and invasion abilities. In addition, HAND2-AS1 rescued enhanced AGS cell proliferation, cell migration, cell invasion abilities and glycolytic process caused by hypoxia via miR-184/HIF3A. Conclusion LncRNA HAND2-AS1 could inhibit GA cell proliferation, migration and invasion abilities and glycolytic process induced by hypoxia through miR-184/HIF3A signaling.

Metastasis and recurrence are major causes of death in gastric cancer patients. Because there are no obvious clinical symptoms during the early stages of metastasis, we sought to isolate highly invasive metastatic gastric cancer cells for future drug screening. We first established a mouse model to observe gastric cancer metastasis in vivo. The incidence of peritoneal metastasis of gastric cancer was much higher than liver or lymph metastasis. Peritoneal metastatic and non-metastatic NUGC-4 cells were isolated from the mouse model. Cell proliferation was measured using CCK-8 assays, while migration and invasion were investigated in Transwell assays. Proteins involved in epithelial-mesenchymal transition were detected by Western blotting. Metastatic gastric carcinoma cells were more proliferative and invasive than primary NUGC-4 cells. The supernatants of metastatic gastric carcinoma cells notably altered the morphology of HMrSV5 peritoneal mesothelial cells and promoted their epithelial-mesenchymal transition. Moreover, primary or metastatic gastric cancer cells co-cultured with HMrSV5 cells markedly increased cancer cell proliferation and invasiveness. Moreover, peritoneal metastatic gastric carcinoma cells in the presence of HMrSV5 cells exhibited most malignant behaviors. Thus, peritoneal metastatic gastric carcinoma cells exhibited high capacities for proliferation and invasion, and could be used as a new drug screening tool for the treatment of advanced gastric cancer and peritoneal metastatic gastric cancer.

Gastric cancer (GC) is a common malignancy with low 5‐year overall survival (OS). Recently, immune therapy has been used to treat cancer. B7H5 and CD28H are novel immune checkpoint molecules. However, the prognostic value of B7H5/CD28H expression in patients with GC remains unclear. In this study, seventy‐one patients diagnosed with GC were included in this study. Patients' GC tissues and matched adjacent tissue constructed a tissue microarray. The expression levels of B7H5 and CD28H were examined using immunohistochemistry. Correlations between the expression of B7H5 and CD28H and the clinical data were evaluated. We found that the expression of B7H5 and CD28H (both P = .001) were higher in GC tumour tissues than in adjacent noncancerous tissues. B7H5/CD28H expression acted as an independent predictive factor in the OS of patients with GC. High expression of B7H5 and CD28H predicted poor outcome. Patients in the B7H5+CD28H+ group had a lower 5‐year OS compared with patients in the B7H5−CD28− group (4.5% vs 55.6%, P = .001). A significant difference was found in the 5‐year OS between patients in the B7H5+CD28H− and B7H5+CD28H+ groups (33.5% vs 4.5%, P = .006). However, there was no correlation between B7H5 and CD28H expression (P = .844). Therefore, B7H5 and CD28H expression are up‐regulated in GC and are independent prognostic factors for overall survival in patients with GC. Although there was no correlation between B7H5 and CD28H expression, high expression of B7H5 and CD28H predicts poor prognosis, especially when both are highly expressed.

Tumor angiogenesis is a key step in the progression of gastric cancer (GC) that delivers essential nutrients and oxygen to tumor cells and distant sites. The cyclic AMP responsive element-binding protein 3-like 4 (CREB3L4) is a transcription factor highly expressed in multiple human cancers. This study aimed to investigate the regulatory effects of CREB3L4 on GC progression and angiogenesis. CREB3L4 was overexpressed in GC tissues and cell lines, and was positively correlated with advanced tumor stage and poor survival in GC patients. The upregulation of CREB3L4 in GC cells increased cell viability, promoted cell proliferation, reduced apoptosis, enhanced cell migration and invasion, and induced the formation of tubule-like endothelial structures, whereas CREB3L4 knockdown impeded tumor cell growth, attenuated cell motility, and prevented human umbilical vein endothelial cells from forming tubule-like structures. In addition, mice inoculated with CREB3L4-deficient GC cells showed significantly suppressed tumor growth compared to the group harboring wild-type tumors. Further analysis revealed that CREB3L4 expression was positively correlated with the level of vascular endothelial growth factor A (VEGFA) in gastric tumors. CREB3L4 regulated the transcription activity of VEGFA by binding to its promoter. The downregulation of VEGFA eliminated CREB3L4-induced GC cell growth and movement, and the formation of endothelial structures; while VEGFA upregulation greatly induced the growth and movement of GC cells with CREB3L4 deficiency. In conclusion, CREB3L4 promoted gastric tumor progression and endothelial angiogenesis by transcriptionally activating the VEGFA promoter, suggesting that therapeutic potential of the CREB3L4/VEGFA axis in GC treatment.

Objective To study the effect of lncRNA HAND2-AS1 on gastric adenocarcinoma (GA) cell property and explore its specific mechanism. Methods Data on stomach adenocarcinoma (STAD) were analyzed to screen differentially expressed lncRNA HAND2-AS1. RNA22-HAS database and dual luciferase reporter assay were applied to confirm the target relationship between HAND2-AS1/HIF3A and miR-184. The HAND2-AS1 and miR-184 expressions in tissue or cells were determined by qRT-PCR and Western blot. Besides, after GA cells (AGS) cultured in normoxic and hypoxic condition, phosphoenolpyruvate (PEP) and lactic acid were quantified by Phosphoenolpyruvate Fluorometric Assay Kit and Lactic Acid Detection kit, respectively. Additionally, colony formation assay, transwell invasion and migration assays were used to evaluate the abilities of cell invasion, migration, and proliferation in distinct conditions. Results The HAND2-AS1 and HIF3A expressions were down-regulated and miR-184 expression was up-regulated in GA tissues and cells. Dual luciferase reporter assay confirmed HAND2-AS1 and HIF3A were targeted by miR-184. AGS cell proliferation abilities were restrained by HAND2-AS1 and HIF3A overexpression and enhanced by miR-184, as well as migration and invasion abilities. In addition, HAND2-AS1 rescued enhanced AGS cell proliferation, cell migration, cell invasion abilities and glycolytic process caused by hypoxia via miR-184/HIF3A. Conclusion LncRNA HAND2-AS1 could inhibit GA cell proliferation, migration and invasion abilities and glycolytic process induced by hypoxia through miR-184/HIF3A signaling.

The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules that participate in extracellular matrix binding, lymphocyte activation, cell migration, and tumor metastasis. In a mouse model of transplant-associated arteriosclerosis, CD44 protein was induced in the neointima of allografted vessels and colocalized with a subset of proliferating vascular smooth muscle cells (SMC). To elucidate the molecular mechanisms regulating CD44 expression in this model, we investigated the regulation of CD44 gene expression by interleukin (IL)-1beta. Treatment of rat aortic SMC with IL-1beta resulted in a 5.3-fold increase in cell surface CD44 expression. Northern analysis showed that IL-1beta promoted a dose- and time-dependent induction of CD44 mRNA which reached 6.6-fold after 48 h, and nuclear run-on analysis showed that IL-1beta increased the rate of CD44 gene transcription within 8 h of stimulation. In transient reporter gene transfection experiments in rat aortic SMC, a 1.4-kilobase fragment of the mouse CD44 5'-flanking sequence mediated this response to IL-1beta. Regulation of CD44 gene expression by the proinflammatory cytokine IL-1beta may contribute to SMC phenotypic modulation in the pathogenesis of arteriosclerosis.

A key manifestation of chronic rejection is an obliterative arteriosclerosis. Myointimal thickening in the vessel is preceded by an endothelialitis involving accumulation of host mononuclear cells in the perivascular and intimal spaces. We report a paratopic LEW-to-F344 rat carotid artery transplantation model developed to study the cells, cytokines, and inflammatory response associated with this early phase of vascular immune injury. Compared with contralateral control arteries and isografts, LEW-to-F344 carotid allografts develop intimal thickening with mononuclear cell infiltration that persists (days 20, 45, 75, 90, and 120). Allografted vessels had dense collections of intimal and adventitial leukocytes (CD45+) consisting of equal numbers of T cells and macrophages. There were small but variable numbers of intimal smooth muscle cells. Intimal cells showed dense staining for tumor necrosis factor-α, interleukin-8, platelet-derived growth factor, iNOS, and ICAM and weaker labeling for interleukin-1β and interleukin-6. There was also prominent staining for interleukin-4 and interleukin-7 with no detectable interferon-γ or interleukin-2 staining and high titer labeling for IgG1 (but not IgG2). The predominance of the T cell infiltrate coupled with interleukin-4 and IgG1 expression in carotid allografts is consistent with a TH2 response. This contrasts with balloon-injured rat carotids, which evoke a macrophage-dependent proliferative response. These findings demonstrate that there are distinct as well as common activation pathways in various forms of vascular injury and the LEW-to-F344 carotid model provides the opportunity to gain insight into molecular mechanisms regulating alloimmune injury in the vessel.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. However, the mechanisms of HHcy-induced arteriosclerosis are largely unknown. In this study, we investigated the role and mechanism of HHcy on vascular remodeling in a carotid arterial vein patch model. We assessed the effect of HHcy on vascular remodeling using a carotid arterial vein patch model in mice with the gene deletion of cystathionine-β-synthase (Cbs, plasma Hcy 310 μM). Vein grafts were harvested 4 weeks after surgery. Cross sections of the transplanted vessel segment were analyzed using Verhoeff-van Gieson staining and Masson’s Trichrome staining for morphological analysis and total collagen protein level assessment. Further, the vessel sections were immunostained with antibodies against α-smooth muscle actin (α-SMA), type 1 collagen, CD45 (leukocyte marker), and Proliferating Cell Nuclear Antigen (PCNA). The effect of Hcy on collagen secretion was examined in cultured rat aortic smooth muscle cells (RASMC) by Western blot analysis. We found that Cbs−/− mice with severe HHcy exhibited thicker neointima and a higher percentage of luminal narrowing in the vein graft. In addition, severe HHcy increased elastin, total collagen, and type 1 collagen deposition in the neointima. Further, severe HHcy increases CD45 positive cells and proliferative cells in the lesions of vein grafts in Cbs−/− mice. Finally, Hcy increases collagen secretion in cultured rat aortic smooth muscle cells (RASMC). These results demonstrate that HHcy increases neointima formation, elastin and collagen deposition following a carotid arterial vein patch. The capacity of Hcy to promote vascular fibrosis and inflammation may contribute to the development of vascular remodeling.

Abstract —Recent studies of mice that lack plasminogen have identified a critical role for this zymogen in arterial remodeling. To permit the use of these (and other) genetically modified mice in the analysis of venous injury, we developed a model in which a patch cut from the external jugular vein of a mouse is grafted to repair a surgically created defect in its carotid artery. In wild-type mice, the venous graft showed initial endothelial denudation and formation of a neointima that progressively and reproducibly expanded in a manner analogous to human vein graft disease, albeit at an accelerated pace. This neointima occupied 37±4.6% of the vessel lumen at day 7 and 66±5.7% at day 20. The proliferative index of neointimal cells assessed by proliferating cell nuclear antigen staining was 50.6±3.6% at day 7 and 15.2±2.0% at day 20. CD45-positive leukocytes and α-actin–positive smooth muscle cells accounted for 9.5±1.0% and 9.9±1.1% of intimal area at day 7, respectively, with the latter increasing to 40.9±2.6% at day 20. Collagen accounted for 6.8±0.7% of intimal area at day 7 and 20.7±1.8% at day 20. Surprisingly, even though arterial neointima formation due to electrostatic and immune-mediated injury is impaired in plasminogen –/– mice, in our study vein graft neointima formation in these mice was not significantly different from that in controls (70.9±6.4 versus 65.6±4.4% luminal occlusion, P =NS). Thus, plasmin proteolysis, although critical in extracellular matrix degradation and cellular migration after arterial injury, does not appear to be so important in vein graft neointima formation, perhaps because of the relative lack of structural barriers to cellular migration in the normal vein wall. This novel model of vein graft injury should be useful for further studies of differences in the response to injury of arterial and venous tissues.

Transplant-associated arteriosclerosis remains an obstacle to long-term graft survival. To determine the contribution to transplant arteriosclerosis of MHC and adhesion molecules from cells of the donor vasculature, we allografted carotid artery loops from six mutant mouse strains into immunocompetent CBA/CaJ recipients. The donor mice were deficient in either MHC I molecules or MHC II molecules, both MHC I and MHC II molecules, the adhesion molecule P-selectin, intercellular adhesion molecule (ICAM)-1, or both P-selectin and ICAM-1. Donor arteries in which ICAM-1, MHC II, or both MHC I and MHC II were absent showed reductions in neointima formation of 52%, 33%, and 38%, respectively, due primarily to a reduction in smooth muscle cell (SMC) accumulation. In P-selectin-deficient donor arteries, neointima formation did not differ from that in controls. In donor arteries lacking both P-selectin and ICAM-1, the size of the neointima was similar to that in those lacking ICAM-1 alone. In contrast, neointima formation increased by 52% in MHC I-deficient donor arteries. The number of CD4-positive T cells increased by 2.8-fold in MHC I-deficient arteries, and that of alpha-actin-positive SMCs by twofold. These observations indicate that ICAM-1 and MHC II molecules expressed in the donor vessel wall may promote transplant-associated arteriosclerosis. MHC I molecules expressed in the donor may have a protective effect.

Background Hypercholesterolemia is thought to be a significant risk factor for coronary vasculopathy in cardiac transplant recipients. Methods and Results We examined the development of arteriosclerosis in mouse carotid artery loops allografted from B.10A(2R) (H-2 h2 ) donors to normocholesterolemic C57BL/6J (H-2 b ) recipients and hypercholesterolemic C57BL/6J recipients in which the apolipoprotein (apo) E gene had been knocked out. Luminal occlusion and cross-sectional neointimal area were greater in arteries allografted into hypercholesterolemic recipients at 15 and 30 days after transplantation. We also measured cellular and extracellular matrix components of the neointima by computerized planimetry of the fractional areas subtended by smooth muscle cells (anti–α-actin stain), collagen (Masson’s trichrome), lipid (oil red O), and leukocytes (anti-CD45). The neointimal area stained for smooth muscle cells was significantly greater in hypercholesterolemic recipients than in normocholesterolemic recipients at 15 and 30 days after allografting. Lipid contributed to neointimal area to a lesser degree, and there was no significant increase in the contribution of collagen or leukocytes. Conclusions Smooth muscle cell accumulation appears to be the principal contributor to the increase in neointimal area observed in arteries allografted into hypercholesterolemic mice.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

To define the role of specific gene deletions and mutations in the development of transplant arteriosclerosis, we generated an accelerated model of the disease in mice. Carotid arteries were transplanted between B.10A(2R) (H-2h2) donor mice and C57BL/6J (H-2b) recipients and compared with arteries isografted between H-2b mice. Immunosuppressive drugs were not used. Within 7 days, the allografted carotid artery formed a neointima composed of mononuclear leukocytes (CD45+) that were predominantly monocytes or macrophages (ie, CD11b+ cells with single-lobed nuclei). CD4+ and CD8+ cells were present as well. By 30 days, the neointima became exuberant, and mononuclear leukocytes were largely replaced by smooth muscle cells. Cells staining for proliferating-cell nuclear antigen were abundantly present in the intima at both early and late time points, indicating the proliferation of mononuclear leukocytes and smooth muscle cells. The area of the intima increased from day 7 to day 30 (P < .0005), as did the number of nuclei (P = .0005), but the density of the nuclei decreased (P = .02), suggesting the formation of extracellular matrix. Six of the eight isografts formed no neointima, and in samples from the remaining two, a single layer of smooth muscle neointimal cells covered just a portion of the vessel circumference. This model, which reproduces many of the features of human transplant arteriosclerosis but at an accelerated pace, should prove useful for determining the roles in transplant arteriosclerosis of genes that code for components of immunologic and inflammatory responses.

Recent gene targeting studies indicate that the plasminogen system is implicated in cell migration and matrix degradation during arterial neointima formation and atherosclerotic aneurysm formation. This study examined whether plasmin proteolysis is involved in accelerated posttransplant arteriosclerosis (graft arterial disease). Donor carotid arteries from wild-type B10.A2R mice were transplanted into either plasminogen wild-type (Plg+/+) or homozygous plasminogen-deficient (Plg-/-) recipient mice with a genetic background of 75% C57BL/6 and 25% 129. Within 15 d after allograft transplantation, leukocytes and macrophages infiltrated the graft intima in Plg+/+ and Plg-/- recipient mice to a similar extent. In Plg+/+ recipients, the elastic laminae in the transplant media exhibited breaks through which macrophages infiltrated before smooth muscle cell proliferation, whereas in Plg-/- recipients, macrophages failed to infiltrate the transplant media which remained structurally more intact. After 45 d of transplantation, a multilayered smooth muscle cell-rich transplant neointima developed in Plg+/+ hosts, in contrast to Plg-/- recipients, in which the transplants contained a smaller intima, predominantly consisting of leukocytes, macrophages, and thrombus. Media necrosis, fragmentation of the elastic laminae, and adventitial remodeling were more pronounced in Plg+/+ than in Plg-/- recipient mice. Expression of the plasminogen activators (PA), urokinase-type PA (u-PA) and tissue-type PA (t-PA), and expression of the matrix metalloproteinases (MMPs), MMP-3, MMP-9, MMP-12, and MMP-13, were significantly increased within 15 d of transplantation when cells actively migrate. These data indicate that plasmin proteolysis plays a major role in allograft arteriosclerosis by mediating elastin degradation, macrophage infiltration, media remodeling, medial smooth muscle cell migration, and formation of a neointima.