Objective— We have previously demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of coronary vasomotion abnormalities, including drug-eluting stent (DES)–induced coronary hyperconstricting responses. Importantly, the adventitia also harbors lymphatic vessels, which may prevent inflammation by transporting extravasated fluid and inflammatory cells. We thus aimed to examine the roles of coronary adventitial lymphatic vessels in the pathogenesis of DES-induced coronary hyperconstricting responses in a porcine model in vivo. Approach and Results— We performed 2 experimental studies. In protocol 1, 15 pigs were divided into 3 groups with or without DES and with bare metal stent. Nonstented sites 20 mm apart from stent implantation also were examined. In the protocol 2, 12 pigs were divided into 2 groups with or without lymphatic vessels ligation followed by DES implantation at 2 weeks later (n=6 each). We performed coronary angiography 4 weeks after DES implantation, followed by immunohistological analysis. In protocol 1, the number and the caliber of lymphatic vessels were greater at only the DES edges after 4 more weeks. In protocol 2, coronary hyperconstricting responses were further enhanced in the lymphatic vessels ligation group associated with adventitial inflammation, Rho-kinase activation, and less adventitial lymphatic vessels formation. Importantly, there were significant correlations among these inflammation-related changes and enhanced coronary vasoconstricting responses. Conclusions— These results provide evidence that cardiac lymphatic vessel dysfunction plays important roles in the pathogenesis of coronary vasoconstrictive responses in pigs in vivo.

Necroptosis is a pivotal process in cancer biology; however, the clinical significance of necroptosis in esophageal squamous cell carcinoma (ESCC) has remained unknown. Therefore, in this study, we aimed to verify the potential involvement of necroptosis in the clinical outcome, chemotherapeutic resistance, and tumor microenvironment of ESCC. Mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunohistochemically examined in 88 surgically resected specimens following neoadjuvant chemotherapy (NAC) and 53 pre-therapeutic biopsy specimens, respectively. Tumor-infiltrating lymphocytes (TILs) were also evaluated by immunolocalizing CD3, CD8, and forkhead box protein 3 (FOXP3) in the residual tumors after NAC. High pMLKL status in the post-NAC resected specimens was significantly correlated with worse prognosis in ESCC patients. Multivariate analysis demonstrated that a high pMLKL status was an independent prognostic factor. In pre-NAC biopsy specimens, a high pMLKL status was significantly associated with a lower therapeutic efficacy. CD8+ TILs were significantly lower in the high-pMLKL group. FOXP3+ TILs were significantly higher in both high-MLKL and high-pMLKL groups. We first demonstrated pMLKL status as an independent prognostic factor in ESCC patients. Our study revealed the possible involvement of necroptosis in the immunosuppressive microenvironment, resulting in the attenuated therapeutic efficacy of NAC and eventual adverse clinical outcomes in ESCC.

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Background Lymph node metastasis is one of the pivotal factors of the clinical outcomes of patients with esophageal cancer receiving neoadjuvant chemoradiation therapy (NACRT). Both the nuclear factor‐erythroid 2‐related factor 2 (Nrf2) signaling pathway and heme oxygenase‐1 (HO‐1) are frequently upregulated in various human malignancies and associated with resistance to chemoradiation therapy, subsequently resulting in adverse clinical outcomes. However, the Nrf2 and HO‐1 status in lymph node metastasis and their differences between primary and metastatic lesions are unknown. Aims To examine the levels of Nrf2 signaling proteins and HO‐1 in primary and metastatic lesions of patients with esophageal squamous cell carcinoma using immunohistochemistry. Methods and Results We immunolocalized Nrf2 signaling proteins in 69 patients with lymph node metastases, who received NACRT with 5‐fluorouracil and cisplatin before esophagectomy. We also compared the findings between primary and metastatic lesions. Residual lymph node metastases were detected in 30 patients and among them, both primary and metastatic lesions were available for evaluation in 25 patients. Subsequently, we correlated the results with patients' survival. Nrf2, HO‐1, and the Ki‐67 labeling index were all significantly lower in the patients with lymph node metastases than in those with primary tumors. Carcinoma cells with high HO‐1 levels were significantly associated with pathological resistance to NACRT. These results suggested that overall and disease‐free survival of esophageal squamous cell carcinoma were significantly associated with both pN2 and high HO‐1 levels, respectively. Conclusions Protein expression in the Nrf2 pathway was significantly lower in patients with lymph node metastases than in those with primary lesions. HO‐1 levels in lymph node metastases could be used to predict the eventual clinical outcome of patients with esophageal cancer receiving NACRT.

Background Thioredoxin reductase 1 (TXNRD1) and heme oxygenase-1 (HO-1) are both involved in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and play key roles in antioxidant responses. In patients with esophageal squamous cell carcinoma (ESCC), the correlation between the expression of these two proteins and the therapeutic response to neoadjuvant chemoradiation therapy (NACRT), as well as the difference in their expression after chemoradiotherapy, remains unknown. Methods Proteins involved in the Nrf2 pathway were immunolocalized in carcinoma cells in ESCC patients on NACRT with 5-fluorouracil and cisplatin, followed by esophagectomy. The 8-hydroxydeoxyguanosine (8-OHdG) levels were used to quantify reactive oxygen species. The changes in immunoreactivity before and after NACRT (Δ) were assessed. Results Tumor reduction following NACRT was significantly attenuated in pre-therapeutic biopsy specimens associated with high HO-1 status. TXNRD1Δ, HO-1Δ, and 8-OHdGΔ were significantly different in the ineffective and effective groups. The overall survival was significantly lower in high Nrf2 and TXNRD1 groups. In addition, high TXNRD1 expression was an independent prognostic factor in the multivariate analysis of overall survival. Conclusions The study findings indicate that HO-1 status in pre-therapeutic biopsy specimens could predict response to NACRT, and TXNRD1 status could predict overall survival of ESCC patients.