ephrinB2 Expression Research Articles

Heterogeneous Kinetics of Nanobubble Ultrasound Contrast Agent and Angiogenic Signaling in Head and Neck Cancer.

Recently developed nanobubble ultrasound contrast agents are a promising tool for imaging and drug delivery in tumors. To better understand their unusual kinetics, we implemented a novel pixel clustering analysis, which provides unique information by accounting for spatial heterogeneity. By combining ultrasound results with proteomics of the imaged tumors, we show that this analysis is highly predictive of protein expression and that specific types of nanobubble time-intensity curve are associated with upregulation of different metabolic pathways. We applied this method to study the effects of two proteins, EphB4 and ephrinB2, which control tumor angiogenesis through bidirectional juxtacrine signaling, in mouse models of head and neck cancer. We show that ephrinB2 expression by endothelial cells and EphB4 expression by cancer cells have similar effects on tumor vasculature, despite sometimes opposite effects on tumor growth. This implicates a cancer-cell-intrinsic effect of EphB4 forward signaling and not angiogenesis in EphB4's action as a tumor suppressor.

EphrinB2-mediated chondrocyte autophagy induces post-traumatic arthritis via rupture of cartilage homeostasis.

EphrinB2, a member of the Ephrin family, has been linked to several orthopaedic conditions. Nevertheless, the correlation between ephrinB2 and post-traumatic arthritis (PTOA) remains unclear. Human PTOA cartilage from human and mouse knee joints was systematically analysed to investigate the relationship between EphrinB2 and PTOA using SO-FG and toluidine blue staining, micro-CT, histomorphometry, immunohistochemistry, immunofluorescence, lentiviral articular injection and insitu end labeling (TUNEL) assays. EphrinB2 expression was significantly downregulated in PTOA chondrocytes. Blocking EphrinB2 increased the breakdown of cartilage matrix in mice with PTOA via reducing the process of chondrocyte autophagy. The presence of severe cartilage damage was evident, as indicated by a considerable decrease in both cartilage thickness and area, accompanied by an increase in chondrocyte death. Altogether, EphrinB2 is required for the maintenance of cartilage homeostasis in post-traumatic arthritis, and EphrinB2 ablation is associated with accelerated chondrocyte matrix degeneration, finally causing damage to the articular cartilage.

Spatiotemporal regulation of dental pulpal innervation in the rat.

The dental pulp is a highly innervated tissue transmitting pain-related sensations in the tooth. Consequently, understanding the intricacies of its innervation mechanism in odontogenesis is crucial for gaining insights into dental pain and developing dental pain-modulating agents. This study examined neuroregulatory molecules such as neurotrophic factors (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], neurotrophin-4 [NTF-4], and neurturin [NRTN]) and neuroinhibitory factors (slit2, ephrin isoforms and netrin-1) in developing rat teeth with follicles. NGF, BDNF and NRTN transcriptions showed time-dependent upregulation, particularly during the root formation stage. In contrast, NTF-4 mRNA was highly expressed at the cap stage, but became downregulated over time. Slit2 and ephrin-B2 expression was distinct at the cap stage and then downregulated in a time-dependent manner. Ephrin-A5 and netrin-1 expression did not significantly change. Immunofluorescence analysis revealed a robust expression of both ephrin-B2 and slit2 in the outer and inner dental epithelia of the enamel organ, a non-neurogenic tissue, during the cap stage of 3rd molar germs. In contrast, BDNF was predominantly localized in dental papilla cells and odontoblasts during the root formation stage. These results suggest that neuroregulatory molecules, such as BDNF, slit2 and ephrin-B2, may be important in identifying therapeutic targets for modulating dental pulp pain.

Improved efficacy of pembrolizumab combined with soluble EphB4-albumin in HPV-negative EphrinB2 positive head neck squamous cell carcinoma.

Patients with relapsed or metastatic head and neck squamous cell carcinoma (HNSCC) after primary local therapy have low response rates with cetuximab, systemic chemotherapy or check point inhibitor therapy. Novel combination therapies with the potential to improve outcomes for patients with HNSCC is an area of high unmet need. This is a phase II single-arm clinical trial of locally advanced or metastatic HNSCC patients treated with a combination of soluble EphB4-human serum albumin (sEphB4-HSA) fusion protein and pembrolizumab after platinum-based chemotherapy with up to 2 prior lines of treatment. The primary endpoints were safety and tolerability and the primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS) and overall survival (OS). HPV status and EphrinB2 expression were evaluated for outcome. Twenty-five patients were enrolled. Median follow up was 40.4 months (range 9.8 - 40.4). There were 6 responders (ORR 24%). There were 5 responders in the 11 HPV-negative and EphrinB2 positive patients, (ORR 45%) with 2 of these patients achieving a complete response (CR). The median PFS in HPV-negative/EphrinB2 positive patients was 3.2 months (95% CI 1.1, 7.3). Median OS in HPV-negative/EphrinB2 positive patients was 10.9 months (95% CI 2.0, 13.7). Hypertension, transaminitis and fatigue were the most common toxicities. The combination of sEphB4-HSA and pembrolizumab has a favorable toxicity profile and favorable activity particularly among HPV-negative EphrinB2 positive patients with HNSCC.

Ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans

Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca2+-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 and these effects were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain.

Predictive value of plasma ephrinB2 levels for amputation risk following endovascular revascularization in peripheral artery disease.

The aim of this study is to investigate the expression levels of ephrinB2 in patients with lower extremity peripheral arterial disease (PAD) and explore its association with the severity of the disease and the risk of amputation after endovascular revascularization. During the period from March 2021 to March 2023, this study collected blood samples and clinical data from 133 patients diagnosed with lower extremity PAD and 51 healthy volunteer donors. The severity of lower extremity PAD patients was classified using the Rutherford categories. The expression of ephrin-B2 in plasma samples was detected using the Western Blotting. Compared to the control group, the levels of serum ephrinB2 in patients were significantly elevated (p < 0.001). Moreover, the plasma EphrinB2 levels were positively correlated with white blood cell counts (r = 0.204, p = 0.018), neutrophil counts (r = 0.174, p = 0.045), and neutrophil-to-lymphocyte ratio (NLR) (r = 0.223, p = 0.009). Furthermore, the AUCs of plasma ephrinB2 level, NLR, and their combination as predictors for amputation events within 30 months after lower extremity PAD endovascular revascularization were 0.659, 0.730 and 0.811. In the high-ephrinB2 group, the incidence of amputation events within 30 months after endovascular revascularization was higher. Plasma EphrinB2 levels may be linked to lower extremity PAD development, inflammation, and postoperative amputation. Combining EphrinB2 and NLR can improve amputation prediction accuracy after endovascular revascularization in lower extremity PAD patients.

Association of ephrinB2 (B2) expression on overall survival (OS) and resistance to PD1/L1 inhibitors (inh) in metastatic urothelial carcinoma (mUC).

4577 Background: B2 is a ligand for EphB4 (B4) which enhances tumor invasion, proliferation, survival and promotes angiogenesis. TCGA data (N= 409) bladder cancer patients (pts) showed B2 high expression (34%) had poor OS vs. low expression (66%) (Hazard Ratio- HR- 0.7 p=0.02). Our phase II trial of sEphB4-HSA, a B2 inhibitor, combined with pembrolizumab in pre-treated mUC (N=70) showed an objective response rate (ORR) of 37%. Pts with high B2 had 52% ORR with 24% complete response (CR) (JCO PMID 35984996). This retrospective study examined the OS and ORR to PD1/L1 inh in pts with mUC and its correlation with B2 expression. Methods: Pts with mUC who received PD1/L1 inh were eligible if they had radiographic response data and tumor tissue for B2 testing. Demographics and disease characteristics were abstracted. Radiographic was assessed using RECIST 1.1. Tumor specimens were analyzed for B2 expression using in-situ hybridization (ISH). Scores of 2-4 were marked High (Hi) and 0-1 Low (Lo). Descriptive statistics summarized the results. Cox Model, logrank and Fisher’s exact test were used for the association of B2 status with OS and ORR, respectively, using SAS 9.4. Results: 143 (N) pts from University of Southern California (USC, n = 49), Dana Farber (DFCI, n = 55), and University of California, Irvine (UCI, n = 39) were included. 101 pts were male (71%). Median age was 73. PD1/L1 inh included pembrolizumab (n = 111, 78%), atezolizumab (n = 25, 17%), nivolumab, avelumab, and durvalumab in 3%, 1%, and 1%, respectively. ORR defined as CR + partial response (PR) was 21% (95% CI 14%, 29%) (N = 136 evaluable). ORR was 12% vs 33% in B2 Hi vs Lo, p = 0.005. The median OS was 17.2 months (mo) (95% CI 13.5, 23.8) (N = 143). Median OS was 14.5 (95% CI 9.4, 21.0) mo vs 24.0 (95% CI 13.7, 60.8) mo for B2 Hi vs Lo, logrank p = 0.022 (HR 1.65 95% CI:1.07, 2.55 Wald p = 0.023). Conclusions: B2 Hi is associated with poor OS and poor ORR in PD1/L1 inh monotherapy treated mUC acting as a biomarker of disease outcome. These data suggest B2-B4 pathway to be a mechanism of resistance to PD1/L1 inh and a therapeutic target. [Table: see text]

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

Hypoxia-Induced Ephrin-B2 Facilitates Proliferation and Induces Glycolytic Metabolism in Cutaneous Squamous Cell Carcinoma by Modulating PKM2/HIF-1α Axis.

Cutaneous squamous cell carcinoma (cSCC) is a fatal disease characterized by metabolic dysregulation. The role of ephrin type-B receptor 2 (ephrin-B2), a crucial molecule in cancer cell biology, in regulating glycolysis and cell proliferation of cSCC is not well understood. This study aimed to investigate the biological pathways by which ephrin-B2 impacts the glycolysis and cell proliferation of cSCC. Ephrin-B2 expression levels in cSCC were determined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Ephrin-B2 expression in cSCC cells was manipulated using overexpression and knockdown approaches. A series of in vitro assays, such as cell counting kit-8 (CCK-8), Transwell assay, immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, and Western blotting, were employed to delineate the biological roles of ephrin-B2/pyruvate kinase muscle isoenzyme 2 (PKM2)/hypoxia-inducible factor 1 alpha (HIF-1α) in proliferation, migration, invasion, and glucose metabolism of cSCC. This study highlights an upregulation of ephrin-B2 expression in cSCC. Knockdown of ephrin-B2 significantly suppressed the proliferation, migration, invasion, and glucose metabolism of cSCC cells. Moreover, ephrin-B2 expression was upregulated under hypoxic conditions. At the molecular level, ephrin-B2 knockdown resulted in the downregulation of PKM2 and HIF-1α expression. Additionally, the overexpression of PKM2 or HIF-1α successfully rescued the diminished proliferation, migration, invasion and glucose metabolism induced by ephrin-B2 knockdown in cSCC cells. These findings suggest that ephrin-B2 suppression may hinder cSCC cell proliferation and glycolytic metabolism, potentially via the PKM2/HIF-1α axis modulation.

ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis.

Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71. In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot. ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts. ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Bioprinted PDLSCs with high-concentration GelMA hydrogels exhibit enhanced osteogenic differentiation in vitro and promote bone regeneration in vivo.

We aimed to explore the osteogenic potential of periodontal ligament stem cells (PDLSCs) in bioprinted methacrylate gelatine (GelMA) hydrogels in vitro and in vivo. PDLSCs in GelMA hydrogels at various concentrations (3%, 5%, and 10%) were bioprinted. The mechanical properties (stiffness, nanostructure, swelling, and degradation properties) of bioprinted constructs and the biological properties (cell viability, proliferation, spreading, osteogenic differentiation, and cell survival in vivo) of PDLSCs in bioprinted constructs were evaluated. Then, the effect of bioprinted constructs on bone regeneration was investigated using a mouse cranial defect model. Ten percent GelMA printed constructs had a higher compression modulus, smaller porosity, lower swelling rate, and lower degradation rate than 3% GelMA. PDLSCs in bioprinted 10% GelMA bioprinted constructs showed lower cell viability, less cell spreading, upregulated osteogenic differentiation in vitro, and lower cell survival in vivo. Moreover, upregulated expression of ephrinB2 and EphB4 protein and their phosphorylated forms were found in PDLSCs in 10% GelMA bioprinted constructs, and inhibition of eprhinB2/EphB4 signalling reversed the enhanced osteogenic differentiation of PDLSCs in 10% GelMA. The in vivo experiment showed that 10% GelMA bioprinted constructs with PDLSCs contributed to more new bone formation than 10% GelMA constructs without PDLSCs and constructs with lower GelMA concentrations. Bioprinted PDLSCs with high-concentrated GelMA hydrogels exhibited enhanced osteogenic differentiation partially through upregulated ephrinB2/EphB4 signalling in vitro and promoted bone regeneration in vivo, which might be more appropriate for future bone regeneration applications. Bone defects are a common clinical oral problem. Our results provide a promising strategy for bone regeneration through bioprinting PDLSCs in GelMA hydrogels.

EphrinB2: Expression of a novel potential target in renal cell carcinoma.

Renal cell carcinoma (RCC) is primarily managed by surgery with the use of systemic targeted therapy in a metastatic setting. Newer targeted therapeutic options are evolving; Eph-ephrin is a potential new pathway. The therapeutic potential of targeting the EphB4-EphrinB2 pathway has been demonstrated in many solid tumors; however, its expression in RCC has only been evaluated in a few studies with limited cases. We herein determine the immunohistochemical expression of EphrinB2 in RCC. A tissue microarray comprising 110 cases of different histological subtypes of RCC and 10 normal kidney tissues were stained with monoclonal anti-EphrinB2 antibody (Abcam, AB201512). The tumor and endothelial cells expressing the EphrinB2 were examined and its expression was correlated with sex, histological subtypes, and tumor nodes metastasis (TNM) stage. Twenty cases of urothelial carcinoma and two unsatisfactory conventional clear cell RCC cases were excluded, and EphrinB2 expression was interpreted in the remaining 88 tumors. EphrinB2 was expressed in 42 out of 88 tumors (47.7%) and was negative in the normal renal parenchyma. There was a statistically significant difference in the expression of EphrinB2 in males (55%) and females (32%). However, no such difference of expression was noted for the histological subtypes and the stages. Half (51%) of Stage 1 (n = 30) and Stage 2 (n = 11) tumors showed EphrinB2 positivity. EphrinB2 is expressed in approximately half of RCC cases. EphrinB2 expression in the early stage cancer might indicate its induction as an early event.

EphrinB2 (B2) expression predicts poor response to PD1/L1 antibodies (Ab) in metastatic urothelial carcinoma (mUC).

4575 Background: B2 is a ligand for EphB4 which enhances tumor invasion, proliferation, survival and promotes angiogenesis. A prior analysis of TCGA data from 409 muscle invasive bladder cancer (MIBC) patients (pts) demonstrated that high (34%) B2 gene expression had poor survival vs. low (66%) expression (Hazard Ratio 0.7 p = 0.02). Our phase II trial of sEphB4-HSA (JCO PMID 35984996) which targets B2, in combination with pembrolizumab in previously treated mUC (N=70) demonstrated an objective response rate (ORR) of 37%. Among pts with high expression of B2, ORR was 52% with 24% complete response (CR). This retrospective study examined the ORR to PD1/L1 Ab in pts with mUC and its correlation with B2 mRNA expression. Methods: Pts with mUC who had received anti PD1/L1 Ab were eligible if they had radiographic response data and tumor tissue for B2 testing. Pts’ demographics and disease characteristics were abstracted. Radiographic images were reviewed using RECIST 1.1. Tumor specimens were analyzed for intensity of B2 expression using in-situ hybridization (ISH). Scores of 2-4 were considered High (Hi) and 0-1 were considered Low (Lo) expression. Standard descriptive statistics were used to summarize study results. Fisher’s exact test was used to test the association of ORR with B2 expression using SAS 9.4. Results: 120 (N) pts from University of Southern California (USC, n=49), Dana Farber (DFCI, n=55), and University of Colorado (UC, n=16) met were included. 88 pts were male (73%). Median age was 72. PD1/L1 Ab were administered in the post-platinum progressive disease (n=95, 79%) and first-line setting (n=25, 21%) and included pembrolizumab (n=88, 73%), atezolizumab (n=26, 22%) and nivolumab, avelumab, and durvalumab, 3%, 1%, and 1%, respectively. Objective response rate (ORR) defined as radiographic CR plus partial response (PR) were calculated and summarized. Among all pts (N=120), ORR was 20% (95% CI 13-28). 62% had B2 Hi expression with a response rate of 12%, and 38% had B2 Lo expression with a response rate of 33%, p=0.009). These proportions were similar for each institution. Conclusions: In addition to conferring poor prognosis in MIBC, B2 expression is associated with poor ORR to PD1/L1 Ab monotherapy in mUC. Given that the combination of B2 inhibition &amp; pembrolizumab was more active in B2 expressing mUC, these data provide further rationale for investigating B2 as a mechanism of resistance to PD1/L1 ab, biomarker of response and a therapeutic target. [Table: see text]

MiR-218-5p-Modified Bone Marrow Mesenchymal Stem Cells Mediate the Healing Effect of EphrinB2-EphB4 Signals on Alveolar Bone Defect

Abnormally expressed miR-218-5p involves in alveolar bone defect. We intend to investigate whether miR-218-5p-modified bone marrow mesenchymal stem cells (BMSCs) mediates the healing effects of EphrinB2-EphB4 signals on the alveolar bone defect. Fifty germ-free rats (6-month-old) were utilized in this study. The grouping was set up as follows: blank group, model group, miR-218-5p group, EphrinB2-EphB4 antagonist group, and positive control group (10 rats in each group). HE staining was employed to quantify bone resorption lacunae number. And the following indicators were monitored: miR-218-5p expression, differentiation status of osteoblasts, concentrations of TNF-α/IL-10/ IL-8, and EphrinB2 and EphB4 expression. As shown in HE staining, massive infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from model group. However, infiltration of inflammatory cells in lesions was moderate in rats from EphrinB2-EphB4 antagonist group and positive control group, which was accompanied by formation of small bone islands. Furthermore, lesser infiltration of inflammatory cells was denoted at the alveolar bone defective sites in rats from the miR-218-5p group, which also exhibited a larger number of newly formed bone trabeculae growing toward the center of lesions. On the 3rd day of culture, absorption lacunae were rare in the model group, while remaining undetectable in other groups. On the 7th day of culture, bone resorption lacunae number in samples from model group was significantly higher in comparison with that in other groups. Meanwhile, it was reduced significantly in miR-218-5p group. However, it was increased in EphrinB2-EphB4 antagonist group and positive control group (P &lt;0.05). An elevation of the intracellular miR-218-5p level was denoted in the modified BMSCs in comparison with those unmodified BMSCs (P &lt; 0.05). In comparison with blank group, other groups exhibited significantly elevated ALP levels, among which model group showed highest level. However, decline of ALP levels was denoted in positive control group, EphrinB2-EphB4 antagonist group and miR-218-5p group, with lowest ALP level in miR-218-5p group (P &lt;0.05). Except blank group, rats in other groups exhibited a significant elevation of TNF-α, IL-10 and IL-8 in the serum, among which those in the model group displayed the most remarkable increase of these cytokines. Rats in miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly reduced levels of IL-8, IL-10 and TNF-α in the serum, with miR-218-5p group showing lowest levels (P &lt; 0.05). In comparison with the blank group, other groups showed significantly enhanced protein expression of EphrinB2 and EphB4, among which the model group displayed the most remarkable enrichment of these proteins. In comparison with the model group, samples from the miR-218-5p group, EphrinB2-EphB4 antagonist group and positive control group exhibited significantly weakened expression of EphrinB2 and EphB4, among which the miR-218-5p group displayed the most remarkable decrease of these proteins (P &lt;0.05). miR-218-5p-modified BMSCs can modulate the EphrinB2-EphB4 signal transduction pathway to produce two-way transmission, which included their inhibition of the osteoclast generation and their enhancement of the osteoclast differentiation. In this way, they aided in alleviating inflammatory response in alveolar bone defective lesions, thereby accelerating the healing process of alveolar bone defect. The function of miR-218-5p-modified BMSCs is mainly achieved in the healing process of the alveolar bone defect.

Notch Signaling-Modified Mesenchymal Stem Cell Patch Improves Left Ventricular Function via Arteriogenesis Induction in a Rat Myocardial Infarction Model

For ischemic cardiomyopathy (ICM) with limited therapeutic options, the induction of arteriogenesis has the potential to improve cardiac function through major restoration of blood flow. We hypothesized that transplantation of a Notch signaling-modified mesenchymal stem cell (SB623 cell) patch would induce angiogenesis and arteriogenesis in ischemic lesions, leading to improvement of left ventricular (LV) function in a rat ICM model. Two weeks after the induction of ischemia, SB623 cell patch transplantation into ICM rats (SB group, n = 10) or a sham operation (no-treatment group, n = 10) was performed. The LV ejection fraction was significantly improved at 6 weeks after SB623 cell patch transplantation (P < 0.001). Histological findings revealed that the number of von Willebrand factor (vWF)-positive capillary vessels (P < 0.01) and alpha smooth muscle actin (αSMA)- and vWF-positive arterioles with a diameter greater than 20 µm (P = 0.002) was significantly increased in the SB group, suggesting the induction of angiogenesis and arteriogenesis. Moreover, rat cardiomyocytes treated with SB623 cell patch transplantation showed upregulation of ephrin-B2 (P = 0.03) and EphB4 (P = 0.01) gene expression, indicating arteriogenesis induction. In conclusion, SB623 cell patch transplantation improved LV function by inducing angiogenesis and arteriogenesis in a rat ICM model.

Progesterone modulates the expression of spinal ephrin-B2 after peripheral nerve injury: New insights into progesterone mechanisms

Recent studies have shown that the ephrin/Eph signaling pathway may contribute to the pathology of neuropathic pain. Drugs like progesterone may be used to counteract both thermal hyperalgesia and mechanical allodynia in different models of neuropathic pain. The present study was designed to determine progesterone’s modulatory role on neuropathic pain and spinal expression of ephrin-B2 following chronic constriction nerve injury (CCI).Thirty-six adult male Wistar rats were used. The sciatic nerve was chronically constricted. Progesterone (5 mg/kg and 15 mg/kg) was administrated for 10 days (from day 1 up to day10) following sciatic constriction. Behavioral tests were performed before surgery (day 0) and on days 1, 3, 7, and 14 after CCI and before progesterone administration on the same days. Western blotting was performed on days 3, 7, and 14th post-surgery. The findings showed that after CCI, the expression of spinal cord ephrin-B2 increased significantly in parallel with mechanical allodynia and thermal hyperalgesia. Post-injury administration of progesterone (15 mg/kg but not 5) decreased mechanical allodynia, thermal hyperalgesia, and the expression of spinal ephrin-B2.It is concluded that post-injury repeated administration of progesterone could be an effective way of alleviating neuropathic pain by suppressing ephrin-B2 activation and helps to make the better design of steroid-based therapies to inhibit pain after peripheral injury.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

Yi Shen Juan Bi Pill alleviates bone destruction in inflammatory arthritis under postmenopausal conditions by regulating ephrinB2 signaling.

Yi Shen Juan Bi Pill (YSJB) is a traditional Chinese medicine (TCM) formulation that has a therapeutic effect upon rheumatoid arthritis (RA), but how YSJB affects bone destruction in arthritis under postmenopausal conditions is not known. We evaluated the therapeutic role of YSJB in bone destruction in postmenopausal arthritis, We used collagen-induced arthritis (CIA) rats who had been ovariectomized (OVX) as models and explored the possible mechanism from the synovium and bone marrow (BM). Arthritis was generated after ovariectomy or sham surgery for 12 weeks. After 14 days of primary immunization, rats were administered YSJB or estradiol valerate (EV) for 28 days. YSJB could prevent bone destruction in the inflamed joints of rats in the OVX + CIA group. CIA promoted osteoclast differentiation significantly in the synovial membrane according to tartrate resistant acid phosphatase (TRACP) staining, and OVX tended to aggravate the inflammatory reaction of CIA rats according to hematoxylin-and-eosin staining. Immunohistochemistry revealed that the synovium did not have significant changes in erythropoietin-producing hepatocellular interactor (ephrin)B2 or erythropoietin-producing hepatocellular (eph) B4 expression after YSJB treatment, but YSJB treatment reduced nuclear factor of activated T cells (NFATc)1 expression. The BM of rats in the OVX + CIA exhibited remarkable increases in the number of osteoclasts and NFATc1 expression, as well as significantly reduced expression of ephrinB2 and ephB4 compared with the CIA group and sham group. YSJB treatment reduced NFATc1 expression significantly but also increased ephrinB2 expression in the BM markedly. These data suggest that YSJB exhibit a bone-protective effect, it may be a promising therapeutic strategy for alleviating bone destruction in arthritis under postmenopausal conditions, and one of the mechanisms is associated with the modulation of ephrinB2 signaling.