G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K⁺ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.Increased arterial constriction and lack of responsiveness to endogenous vasodilators is a hallmark of vascular disease leading to poor health prognosis. Defining the factors that determine vascular smooth muscle (VSM) activity and modulation by vasorelaxant molecules is therefore imperative for a better understanding of vascular disease. Potassium channels are key regulators of VSM tone because they promote membrane hyperpolarization that limits the activity of voltage-dependent calcium channels known to precipitate vasoconstriction (1). The Kv7 family of voltage-dependent potassium channels and the Kv7.4 isoform, in particular, has a fundamental role in this process. There are five Kv7 isoforms (Kv7.1–Kv7.5) of which Kv7.1, Kv7.4, and Kv7.5 are consistently expressed within VSM, where the predominant molecular architecture is a Kv7.4/Kv7.5 heterotetramer (2, 3). Activation of Kv7 channels produces relaxation of numerous arteries (4–8), whereas blockade of Kv7 channels results in contraction of vessels at rest (7, 9–11) or an inhibition of endogenously derived vasorelaxations (2, 11–13). In addition, molecular reduction of Kv7.4 reduces responses to various Gs-coupled vasodilators in a number of arteries (2, 11). Crucially, Kv7.4 abundance is reduced in various arteries from hypertensive animals (6, 11, 12) where relaxant responses to endogenous vasodilators are also impaired (11, 12). Despite the key role of Kv7.4 channels in the regulation of VSM, and their involvement in mediating Gs-coupled vasodilator responses, the factors that regulate channel activity are poorly understood, and the signals linking Kv7.4 to Gs-receptor activation remain to be elucidated.G-protein–coupled receptor (GPCR) activation promotes the exchange of GDP for GTP resulting in disassociation of the heterotrimeric Gαβγ complex from the receptor into Gα-GTP and Gβγ (14). It is now established that the Gβγ complex as well as the Gα–GTP activates various intracellular signaling pathways (see refs. 15, 16 for reviews). Gβγ subunits also modulate various ion channels directly, a phenomenon of which there are only a handful of examples, with the positive regulation of an inwardly rectifying K⁺ channel in the heart the best characterized (17, 18). In this study, we explored whether Gβγ subunits modulated Kv7.4 channels and therefore function as signaling intermediates following receptor stimulation. Our results show that not only are Gβγ subunits able to enhance Kv7 channels, but also that they are a crucial requirement for the basal activity of the Kv7.4 channel.     

Wnt/β‐catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin‐embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine‐specific Wnt signaling molecule mRNA expression levels were evaluated by two‐step real‐time qPCR. Spatial tissue protein expression of β‐catenin, Wnt‐11, Wnt‐4, and Dkk‐1 was determined by immunohistochemistry. There was significantly decreased Wnt‐11 and increased β‐catenin, Wnt‐5b, Dkk‐1, Lrp6, Wif‐1, Axin1, and SC‐PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β‐catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1433–1438, 2015.     

Can we afford to ignore the biology of joint healing and graft incorporation after ACL reconstruction?

Anterior cruciate ligament (ACL) reconstruction is successful at restoring stability to return ACL injured patients to high-demand work, sports, and recreational activities. The development of posttraumatic osteoarthritis (OA) in roughly half of patients just 10–15 years after ACLR highlight the need to improve clinical care pathways. Graft failure and reinjury rates, which further increase OA risk, also remain high for younger and more active patients. The biological components of joint recovery and graft incorporation, therefore, impact short- and long-term clinical outcomes. Biochemical and magnetic resonance imaging (MRI) data show substantial compromise of articular cartilage metabolism and matrix composition after ACL injury and reconstructive surgery suggesting a potential need for activity modulation in early recovery. Furthermore, joint recovery is variable with compositional MRI studies showing progressive cartilage degeneration 1 and 2 years after ACLR. Biopsy and MRI studies also show high variability in ACL graft characteristics within the 1st year after ACLR followed by continued graft maturation into the 2nd year and beyond. To improve the care of ACL injured patients, there is a critical need for clinical attention and scientific inquiry into timing the reintroduction of higher load activities in relationship to neuromuscular recovery, joint biology, and graft maturation. In addition to symptomatic and mechanical recovery, development and validation of biological markers for joint and cartilage homeostasis as well as ACL graft healing are needed for personalized decision making on rehabilitation needs, reduction of OA risk, and resumption of athletic, recreational, and vocational activities.     

乳腺癌新辅助化疗前后肿瘤相关巨噬细胞相关基因变化的生物信息学分析

背景与目的 乳腺癌是全球女性发病率最高的恶性肿瘤,化疗是乳腺癌最重要的治疗方式之一,最近的研究表明,化疗可能通过增强肿瘤微环境中的抗肿瘤免疫力来发挥抗肿瘤效应。因此,本研究通过生物信息学分析明确乳腺癌患者新辅助化疗（NAC）前后肿瘤相关巨噬细胞（TAMs）及相关基因的变化,评估NAC对乳腺癌患者免疫影响。方法 GEO数据库输入“Breast Cancer”,“TAMs”,“Chemotherapy”进行检索,选择人乳腺癌组织的GSE134600数据集进行分析。通过R包（limma函数）筛选乳腺癌患者NAC前后组织样本中差异表达基因（DEGs）。对所有DEGs进行GO功能富集和KEGG通路分析。通过Cytoscape软件对DEGs进行蛋白互作网络可视化,并筛选关键核心基因,通过cBioPortal对10个关键基因进行突变分析。使用R包（CIBERSORT）对GSE134600数据中的免疫细胞分布及相关性进行评估。结果 鉴定出751个乳腺癌NAC前后DEGs（409个上调基因和342个下调基因）。通过GO富集分析DEGs的生物过程（BP）、细胞组分（CC）和分子功能（MF）。在BP中主要富集在I型干扰素（IFN-I）信号通路/病毒应答与防御和病毒生命周期方面;在CC中主要富集在细胞膜的外在成分和细胞膜的细胞质侧方面;在MF中主要富集在细胞因子受体结合、双链RNA结合和脂肽结合方面。KEGG通路富集分析中,DEGs主要富集在甲型H1N1流感、麻疹、丙型肝炎、冠状病毒病COVID-19、NF-κB信号通路、EBV病毒感染、NOD样受体信号通路和阿米巴病信号通路。通过CytoHubba插件筛选出乳腺癌NAC前后TAMs相互作用程度最高的前10个关键基因：IFIT1、ISG15、MX1、MX2、IRF7、RSAD2、IFIT3、IFI35、IFI6、IFITM1。多组学分析发现IFIT1、MX1和MX2主要发生缺失突变,IFIT1主要发生基因深度删除,而MX1和MX2主要发生基因扩增。NAC后乳腺癌组织中M0巨噬细胞、CD8⁺T细胞及M2巨噬细胞含量减少,M0巨噬细胞与记忆性B细胞成正相关（r=0.64）,与未活化的CD4⁺记忆性T细胞呈负相关（r=-0.66）。结论 所发现的乳腺癌患者NAC前后TAMs相关的DEGs与干扰素信号通路密切相关,提示干扰素信号通路在NAC可能通过改变TAMs而发挥重要作用。同时NAC前后M0巨噬细胞发生明显改变,提示化疗可能通过改变M0巨噬细胞分布及免疫功能调节对肿瘤的免疫应答。     

Extracellular nucleotide signaling in solid organ transplantation

The role of extracellular purine nucleotides, including adenosine triphosphate (ATP) and adenosine, as modulators of posttransplantation outcome and ischemia‐reperfusion injury is becoming increasingly evident. Upon pathological release of ATP, binding and activation of P2 purinergic surface receptors promote tissue injury and inflammation, while the expression and activation of P1 receptors for adenosine have been shown to attenuate inflammation and limit ischemia‐induced damage, which are central to the viability and long‐term success of allografts. Here we review the current state of the transplant field with respect to the role of extracellular nucleotide signaling, with a focus on the sources and functions of extracellular ATP. The connection between ischemia reperfusion, purinergic signaling, and graft preservation, as well as the role of ATP and adenosine as driving factors in the promotion and suppression of posttransplant inflammation and allograft rejection, are discussed. We also examine novel therapeutic approaches that take advantage of the ischemia‐reperfusion‐responsive and immunomodulatory roles for purinergic signaling with the goal of enhancing graft viability, attenuating posttransplant inflammation, and minimizing complications including rejection, graft failure, and associated comorbidities.     

Trained immunity in organ transplantation

Consistent induction of donor‐specific unresponsiveness in the absence of continuous immunosuppressive therapy and toxic effects remains a difficult task in clinical organ transplantation. Transplant immunologists have developed numerous experimental treatments that target antigen‐presentation (signal 1), costimulation (signal 2), and cytokine production (signal 3) to establish transplantation tolerance. While promising results have been obtained using therapeutic approaches that predominantly target the adaptive immune response, the long‐term graft survival rates remain suboptimal. This suggests the existence of unrecognized allograft rejection mechanisms that contribute to organ failure. We postulate that trained immunity stimulatory pathways are critical to the immune response that mediates graft loss. Trained immunity is a recently discovered functional program of the innate immune system, which is characterized by nonpermanent epigenetic and metabolic reprogramming of macrophages. Since trained macrophages upregulate costimulatory molecules (signal 2) and produce pro‐inflammatory cytokines (signal 3), they contribute to potent graft reactive immune responses and organ transplant rejection. In this review, we summarize the detrimental effects of trained immunity in the context of organ transplantation and describe pathways that induce macrophage training associated with graft rejection.     

A randomized,phase 1b study of the pharmacokinetics,pharmacodynamics, safety,and tolerability of bleselumab,a fully human,anti‐CD40 monoclonal antibody,in kidney transplantation

This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of various doses of the anti‐CD40 monoclonal antibody bleselumab (ASKP1240) in de novo kidney transplant recipients receiving concomitant standard immunosuppression over 90 days posttransplant. Transplant recipients were randomized (1:1:1:1:1) to bleselumab 50 mg, 100 mg, 200 mg, or 500 mg, or placebo, in addition to standard maintenance immunosuppression. The primary pharmacokinetic endpoints were AUC_inf, C_max, and AUC_last. The primary pharmacodynamic endpoint was B cell CD40 receptor occupancy over time. Overall, 50 kidney transplant recipients were randomized; 45 received their randomized treatment (bleselumab [n = 37] or placebo [n = 8]). AUC_inf and AUC_last demonstrated a more than dose‐proportional increase in the range of 50‐500 mg, and C_max increased linearly with increasing dose. Maximal receptor occupancy for B cell CD40 was reached at all dose levels and was prolonged as dose increased. No kidney transplant recipients experienced cytokine release syndrome or a thromboembolic event. Treatment‐emergent anti‐bleselumab antibodies were found in one kidney transplant recipient in the bleselumab 50 mg group; these were detected only at Day 7. Overall, bleselumab demonstrated nonlinear pharmacokinetics and dose‐dependent prolonged B cell CD40 receptor occupancy and was well tolerated at all doses (ClinicalTrials.gov: NCT01279538).     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

Live birth after cervical ectopic uterus transplantation in mice

An ideal animal model is a prerequisite for the basic research of uterus transplantation. This study aimed to develop a new cervical ectopic uterus transplantation mice model, which was established by vascular anastomosis of the right common iliac artery and vein of the donor with the right common carotid artery and external jugular vein of the recipient, respectively, using the cuff method. The survival status of the transplanted uterus was assessed by macroscopic observation and histological examination after surgery, and the function of the graft uterus was tested by verifying whether the pregnancy is possible. A total of 40 transplants were performed, of which only 1 failed due to donor hemorrhage. After 26 transplants, the total operation time reduced to 52.4 ± 3.8 minutes, of which the total ischemia time took 6.6 ± 1.1 minutes. Sixty days after transplantation, all the graft uteri had a good blood supply and spontaneous contraction. The histology showed no significant difference between the transplanted uterus and the native. Embryo transfer experiments have proven that the transplanted uterus has uterine function. In conclusion, this new model is an effective and simple mice model for the studies of the scientific issues related to uterus transplantation.