Connexin 43 Regulates Intercellular Mitochondrial Transfer from Human Mesenchymal Stromal Cells to Chondrocytes.

Background: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin43 (Cx43, encoded by GJA1 ) and the truncated internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Methods: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1+ and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 hrs in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Results: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Conclusions: Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.

Software BreastAnalyser for the semi-automatic analysis of breast cancer immunohistochemical images.

Breast cancer is the most diagnosed cancer worldwide and represents the fifth cause of cancer mortality globally. It is a highly heterogeneous disease, that comprises various molecular subtypes, often diagnosed by immunohistochemistry. This technique is widely employed in basic, translational and pathological anatomy research, where it can support the oncological diagnosis, therapeutic decisions and biomarker discovery. Nevertheless, its evaluation is often qualitative, raising the need for accurate quantitation methodologies. We present the software BreastAnalyser, a valuable and reliable tool to automatically measure the area of 3,3'-diaminobenzidine tetrahydrocholoride (DAB)-brown-stained proteins detected by immunohistochemistry. BreastAnalyser also automatically counts cell nuclei and classifies them according to their DAB-brown-staining level. This is performed using sophisticated segmentation algorithms that consider intrinsic image variability and save image normalization time. BreastAnalyser has a clean, friendly and intuitive interface that allows to supervise the quantitations performed by the user, to annotate images and to unify the experts' criteria. BreastAnalyser was validated in representative human breast cancer immunohistochemistry images detecting various antigens. According to the automatic processing, the DAB-brown area was almost perfectly recognized, being the average difference between true and computer DAB-brown percentage lower than 0.7 points for all sets. The detection of nuclei allowed proper cell density relativization of the brown signal for comparison purposes between the different patients. BreastAnalyser obtained a score of 85.5 using the system usability scale questionnaire, which means that the tool is perceived as excellent by the experts. In the biomedical context, the connexin43 (Cx43) protein was found to be significantly downregulated in human core needle invasive breast cancer samples when compared to normal breast, with a trend to decrease as the subtype malignancy increased. Higher Cx43 protein levels were significantly associated to lower cancer recurrence risk in Oncotype DX-tested luminal B HER2- breast cancer tissues. BreastAnalyser and the annotated images are publically available https://citius.usc.es/transferencia/software/breastanalyser for research purposes.

Incarcerated Men: Pain Experiences Shaped by Altered Independence and Loss of Autonomy.

BACKGROUND: Individuals experiencing pain while incarcerated depend on nurses, security structures, processes, and regulations for relief. PURPOSE: The intent of this research was to understand men's experience of pain during incarceration to inform correctional nursing practice. METHOD: Interpretive description, co-positioned with relational ethics, guided this study. Twelve incarcerated men participated in a single 1-hour interview that was audio-recorded and transcribed. The resultant text was analyzed for themes. FINDINGS: The two main themes were dependence on staff and institutional processes and dependence on oneself and others who were incarcerated. Participants reported a substantial loss in their ability to access pain-relieving medications, products, and services. Unresponsive or delayed pain-relieving interventions from nurses contributed to their sense of indignity, disrespect, and injustice. The participants employed whatever was available, mainly exercise equipment and social support, to manage their pain. DISCUSSION: Nurses must engage with incarcerated patients meaningfully to understand and respond to contextual factors that influence their pain experiences. Participants identified loss of autonomy and dependence on nurses as barriers to their pain relief. CONCLUSION: Nurse engagement and responsiveness are crucial to altering incarcerated men's perceptions of injustice or helplessness while improving their pain experiences. Nurses must also foster autonomous pain-management approaches that men can use without limitations within correctional settings.

Connexin 43 in Dermatofibroma and Dermatofibrosarcoma Protuberans: Diagnostic, Pathogenic, and Therapeutic Implications.

ABSTRACT: Connexins play a crucial role in the formation of gap junctions that connect cells to each other, as well as cells to the surrounding environment. In recent years, connexin 43 has been extensively studied in various human tumors. In this study, we conducted an immunohistochemical analysis to evaluate the expression of connexin in 16 dermatofibromas (DFs) and 13 dermatofibrosarcoma protuberans (DFSP). Connexin was diffusely expressed in the cytoplasm of all DFs with moderate or strong intensity, whereas all DFSPs showed negative staining. In addition to its diagnostic implications, the loss of Cx43 may elucidate the invasive capacity of DFSP and offer a potential avenue for future therapeutic interventions.

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases.

Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of 10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10Panx1). In adhesion assays, a "double-stapled" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease.

Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1.

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure-activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1's channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Quantitative PET tracking of intra-articularly administered 89Zr-peptide-decorated nanoemulsions.

Intra-articular (IA) administration of drugs for the treatment of diseases such as rheumatoid arthritis, osteoarthritis and psoriatic arthritis is a common strategy; however, the rapid clearance from the synovial fluid restricts their effectivity due to the limited retention time. Drug Delivery Systems (DDS) are currently being developed to increase their joint retention time. This study compares the biodistribution and retention time of a senolytic peptide (PEP), with potential application in osteoarthritis disease, and this senolytic peptide encapsulated in a DDS based on a lipid nanoemulsion (PEPNE) by using positron emission tomography (PET) imaging. To this aim, the PEP was conjugated with a chelating agent (DFO) and radiolabeled with zirconium-89 (89Zr). Then, [89Zr]-PEP was encapsulated in a novel nanoemulsion formulation, composed by vitamin E, sphingomyelin, and a lipid-PEG. Afterward, healthy rats were administered with either the [89Zr]-PEP or the [89Zr]-PEP-NE via IA injection and underwent PET scans at 0.5-, 24-, 48-, 72-, 168-, 240- and 336 h post-injection. To assess the biodistribution of both radiotracers, several volume-of-interest were manually drawn in different organs of the rat body and the %ID/organ was calculated. The [89Zr]-PEP was successfully encapsulated in the NE and their physicochemical properties were minimally affected by the radiolabeling buffer. Adequate stability of both [89Zr]-PEP and [89Zr]-PEP-NE was found in synovial fluid over 72 h. Quantitative data from PET images revealed a significantly higher [89Zr]-PEP-NE retention in the injected knee than with [89Zr]-PEP in all follow-up PET scans. The [89Zr]-PEP %ID/organ values in the liver and kidney were significantly higher than those from [89Zr]-PEP-NE, which might indicate a faster elimination of the [89Zr]-PEP. Therefore, the study highlights the higher retention time on the target site of the [89Zr]-PEP-NE which may improve the therapeutic effects of the peptide. Thereby, the novel nanoemulsion formulation seems to be a successful DDS for IA injection. In addition, these results represent the first study that evaluates the distribution of a PET-guided DDS after its IA administration.

Extracellular vesicles enriched in connexin 43 promote a senescent phenotype in bone and synovial cells contributing to osteoarthritis progression.

The accumulation of senescent cells is a key characteristic of aging, leading to the progression of age-related diseases such as osteoarthritis (OA). Previous data from our laboratory has demonstrated that high levels of the transmembrane protein connexin 43 (Cx43) are associated with a senescent phenotype in chondrocytes from osteoarthritic cartilage. OA has been reclassified as a musculoskeletal disease characterized by the breakdown of the articular cartilage affecting the whole joint, subchondral bone, synovium, ligaments, tendons and muscles. However, the mechanisms that contribute to the spread of pathogenic factors throughout the joint tissues are still unknown. Here, we show for the first time that small extracellular vesicles (sEVs) released by human OA-derived chondrocytes contain high levels of Cx43 and induce a senescent phenotype in targeted chondrocytes, synovial and bone cells contributing to the formation of an inflammatory and degenerative joint environment by the secretion of senescence-associated secretory associated phenotype (SASP) molecules, including IL-1ß and IL-6 and MMPs. The enrichment of Cx43 changes the protein profile and activity of the secreted sEVs. Our results indicate a dual role for sEVs containing Cx43 inducing senescence and activating cellular plasticity in target cells mediated by NF-kß and the extracellular signal-regulated kinase 1/2 (ERK1/2), inducing epithelial-to-mesenchymal transition (EMT) signalling programme and contributing to the loss of the fully differentiated phenotype. Our results demonstrated that Cx43-sEVs released by OA-derived chondrocytes spread senescence, inflammation and reprogramming factors involved in wound healing failure to neighbouring tissues, contributing to the progression of the disease among cartilage, synovium, and bone and probably from one joint to another. These results highlight the importance for future studies to consider sEVs positive for Cx43 as a new biomarker of disease progression and new target to treat OA.

Connexin 43 Expression in Cutaneous Biopsies of Lupus Erythematosus.

INTRODUCTION: Gap junctions are channels between adjacent cells formed by connexins (Cxs). Cxs also form hemichannels that connect the cell with its extracellular milieu. These channels allow the transport of ions, metabolites, and small molecules; therefore, Cxs, and more specifically, connexin (Cx) 43 has been demonstrated to be in control of several crucial events such as inflammation and cell death. MATERIAL AND METHODS: We examined the immunostaining of Cx43 in the endothelia of the cutaneous blood vessels of biopsies from 28 patients with several variants of lupus erythematosus. RESULTS: In 19 cases (67.86%), staining of more than half of the dermal vessels including both vessels of the papillary and of the reticular dermis was identified. Only in 4 cases (14.28%), less than 25% of the vessels in the biopsy showed expression of the marker. CONCLUSIONS: Our results suggest a role of Cx43 in regulating the endothelial activity in lupus erythematosus, which also opens a door for targeted therapeutic options.

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence.

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

Toward integrated pediatric medicine: Key learnings from the pediatric integrative medicine trial.

OBJECTIVE: The purpose of this study is to explore the facilitators to integrating complementary therapies in conventional pediatric hospital practice based on the experiences of parents, healthcare providers, and complementary therapy providers. DESIGN: This study is part of a larger research study that examined the introduction of a pediatric integrative medicine service in an acute care children's hospital in Canada. A qualitative descriptive study was conducted using semi-structured one-on-one telephone and in-person interviews with a sample of parents of children, as well as healthcare providers and complementary therapy providers. RESULTS: A total of 50 individuals, from key-stakeholder groups, were interviewed between May 2014 and January 2016. This study identified the following facilitators for the integration of complementary therapies within conventional care: 1) stakeholders' open-mindedness and familiarity with care practices outside of their experiences; 2) stakeholders' open communication, respect for eachothers' roles in the process of care, and appreciation for the role of complementary therapies within conventional medicine; and 3) stakeholders' receptiveness to redefining the meaning of a 'positive outcome' in the context of hospital care. CONCLUSION: The findings of this study demonstrate that some of the existing barriers to the integration of complementary therapies in conventional hospital care could be mediated by creating an environment where the fundamental value of commitment to patient wellbeing is equally shared by all stakeholders.

A caregiver, an expert, a patient: How complementary therapies support the roles of parents of children with life threatening conditions in hospital settings.

OBJECTIVE: The purpose of this study was to understand emerging roles of parents of hospitalized children with life threatening conditions and to explore how complementary therapies integrated into conventional pediatric care may shift and/or support these roles. DESIGN: This study is part of a larger research study that examined the introduction of a pediatric integrative medicine service at an acute care children's hospital in Canada. A qualitative descriptive study was conducted using one-on-one telephone interviews with a sample of parents of children included in the larger study. Children had access to complementary therapies including Reiki, massage therapy, and acupuncture. RESULTS: A total of 36 interviews were conducted between May 2014 and January 2016. This study found that parents of hospitalized children assume complex roles including that of caregiver, expert and patient (due to high levels of stress and anxiety). Moreover, the study reveals that the integration of complementary therapies with conventional care supports these parental roles. CONCLUSION: This study reveals that complementary therapies, introduced as a part of integrated approach to pediatric hospital care, and aimed primarily at managing distressing symptoms in patients, had simultaneously a positive contribution in providing parents with the means to navigate the complexities of parenting in the pediatric oncology and cardiology hospital wards and addressing some of their own needs.

Expression of connexin 43 by atypical fibroxanthoma.

INTRODUCTION: Connexins are transmembrane channel proteins that interconnect adjacent cells and allow the exchange of signaling molecules between cells and the extracellular milieu. They have been investigated in many tumors to obtain information about tumor nature, behavior, and prognosis. METHODS: Herein, we present a study on the immunohistochemical expression of connexin (Cx) 43 in 16 cases of atypical fibroxanthoma (AFX). For the immunohistochemical staining, a tissue array was obtained from the paraffin-embedded blocks. RESULTS: The expression was membranous and cytoplasmic in all cases. Thirteen cases (81.25%) showed strong staining. In the other three cases (18.75%), the staining was medium. None of the cases showed nuclear staining. Fifteen out of 16 cases showed a diffuse pattern, and only one case showed a focal pattern. CONCLUSIONS: Our results suggest that Cx43 may play an important role in the natural behavior of AFX.

Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis.

Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.

Walking together: women with the severe symptoms of menopause propose a platform for a walking program; outcome from focus groups.

BACKGROUND: Menopause and midlife are stages in a woman's life that can be marked by debilitating symptoms and increasing risks for cancer, cardiovascular, metabolic, and bone health issues. Walking represents a simple, low cost, and widely accessible activity with proven health benefits, though its therapeutic effect on alleviating menopause symptoms is not well characterized. Women are generally not opposed to exercise programs; however, increasing or maintaining exercise levels remains a challenge. We undertook a qualitative descriptive study to explore features of a walking program that would be conductive to menopausal women's participation, as well as to inform the development of such a program. METHODS: We conducted focus groups with women recruited from two menopause clinics and who suffered from moderate to severe menopause symptoms. The focus groups were audio recorded and transcribed. Women were prompted to talk about their menopause experience and exercise practice and how they would envision a walking exercise program that would keep them engaged. Qualitative content analysis was used to analyze the data and to identify characteristics of a walking exercise program. RESULTS: Twenty women participated in 5 focus groups. Women were very interested in trying walking as a means of staying healthy and possibly reducing menopause symptoms. Four major characteristics emerged as important for a walking program: (a) sensitivity to health realities of menopausal women, (b) inclusivity of various needs/levels of physical ability, (c) attentiveness to the need for mutual social support, (d) flexibility in planning of locations and scheduling. A restricted social network platform with features catering to women in menopause was suggested as suitable to initiate and sustain an adequate walking program. CONCLUSIONS: The findings of this study will be essential in designing a program that would be attractive for women to start and maintain a walking habit. The program would assist in elucidating whether walking is a useful and valuable alternative therapy for menopausal symptoms and, ultimately, might help women staying fit in midlife and postmenopausal.

Emerging functions and clinical prospects of connexins and pannexins in melanoma.

Cellular communication through gap junctions and hemichannels formed by connexins and through channels made by pannexins allows for metabolic cooperation and control of cellular activity and signalling. These channel proteins have been described to be tumour suppressors that regulate features such as cell death, proliferation and differentiation. However, they display cancer type-dependent and stage-dependent functions and may facilitate tumour progression through junctional and non-junctional pathways. The accumulated knowledge and emerging strategies to target connexins and pannexins are providing novel clinical opportunities for the treatment of cancer. Here, we provide an updated overview of the role of connexins and pannexins in malignant melanoma. We discuss how targeting of these channel proteins may be used to potentiate antitumour effects in therapeutic settings, including through improved immune-mediated tumour elimination.

New Therapeutic Strategies for Osteoarthritis by Targeting Sialic Acid Receptors.

Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing α-2,6-linked sialic acids to those that contain α-2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of α-2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the α-2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting α-2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.

Expression of Connexin 43 in 32 Cases of Merkel Cell Carcinoma.

INTRODUCTION: Connexins (Cxs) are channel proteins that allow direct connection among cells and between cells and the extracellular space. There is very little information in the literature on the expression of Cxs by Merkel cell carcinoma (MCC). MATERIALS AND METHODS: Thirty-two cases of MCC were recovered from our archives and studied immunohistochemically for Cx43. RESULTS: All our cases expressed several neuroendocrine markers. Most cases showed nonimmunohistochemically perceptible staining for Cx43. There was no difference between Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative cases. One case could not be evaluated. Only 2 cases showed a focal (10% of the tumor) membranous staining of Cx43. One of these cases was MCPyV-negative and, in the other, CM2B4 could not be evaluated. CM2B4 was positive in 18 cases and negative in 13 cases, and it could not be evaluated in 1 case. CONCLUSIONS: MCC shows a low Cx43 level, with no differences between MCPyV-positive and MCPyV-negative cases. Therefore, this opens the door for Cx43 targeting in therapeutic approaches to MCC.