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Yes / Since the 1980's, changes in nursing education have inadvertently led to diminishing anatomy and physiology content in curricula (Taylor et al., 2015). The need for nurses to have a thorough grounding in these subjects is undisputed; however, the pedagogical principles for anatomy and physiology education have been under scrutiny (Perkins, 2019). Anatomy and physiology are typically incorporated as part of bioscience, which also encompasses genetics, microbiology, pharmacology, and pathophysiology (Horiuchi-Hirose et al., 2023). Registered nurses and nursing students often express anxiety about studying bioscience and its perceived difficulty, largely due to difficulties in applying theory to practice (Craft et al., 2013, Craft et al., 2017, Meedya et al., 2019). Despite this, there remains a recognition that bioscience knowledge is important for effective nursing practice (Danielson and Berntsson, 2007, Horiuchi-Hirose et al., 2023). / The full-text of this article will be released for public view at the end of the publisher embargo on 15 Dec 2024.

Pulmonary arterial hypertension (PAH) is a progressive and debilitating disease characterised by increases in pulmonary vasoconstriction and excessive remodelling of the pulmonary arteries. Together, these processes lead to sustained elevations in pulmonary arterial pressure, right heart failure and eventual death if left untreated. Despite the number and variety of treatment options available, the survival rate in incident and prevalent cases of PAH remains poor. Therefore, a better understanding of the pathobiology of PAH is required to generate novel therapeutic approaches with improved efficiency in patients. In PAH there is a well described gender bias. Women are consistently reported to represent up to 75% of the total PAH population; however, the reasons for this female predominance remain unclear. Recently, estrogen has been implicated as a major risk factor, for example, elevated estrogen levels and alterations in estrogen metabolism are closely correlated with PAH development in females. The role of testosterone in PAH is currently under investigated. Effects of estrogen are mediated through two classical estrogen receptors (ER)-α and –β, or the novel G-protein-coupled estrogen receptor (GPER). Expression of all of these receptors is identified in pulmonary vasculature, including in smooth muscle and endothelial cells. The role they play in PAH pathogenesis in females is largely undetermined. Given the diverse effects of estrogen described in the pulmonary vasculature during PAH, for example, proliferative effects in pulmonary artery smooth muscle cells (PASMCs), we hypothesised that estrogen receptors play an integral role in PAH in females. To examine this, we used both translational and experimental studies to characterise ERs in PAH. Chronic hypoxic male and female mice, and mice over-expressing the serotonin transporter (SERT+ mice), which demonstrate female susceptibility, were used to investigate the effects of an ERα antagonist in vivo. GPER knockout mice were also investigated in chronic hypoxia. In situ and in vitro studies in human PASMCs with ER agonists and antagonists added clinical relevance to our findings. In addition, testosterone manipulation was investigated in male mice by castration in vivo. Immunohistochemistry, immunoblotting and qRT-PCR analysis demonstrated that ERα was increased in PASMCs and pulmonary arteries from female PAH patients and chronic hypoxic mice, respectively. On the other hand, ERβ was decreased in PAH and hypoxia. It was also observed that females expressed higher levels of ERα in PAH compared to males whereas ERβ was lower in females. PAH was assessed by measuring right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling and muscularisation. Chronic hypoxia induced-pulmonary hypertension (PH) was attenuated in female mice dosed with the ERα antagonist MPP, shown by marked reductions in RVSP and pulmonary vascular remodelling. Hypoxic male mice remained unaffected by MPP treatment. Spontaneous PH and chronic hypoxia induced-PH observed in female SERT+ mice were reversed by treatment with MPP. Immunoblotting and qRT-PCR analysis revealed that the possible mechanism involved in the beneficial effect of MPP in females in vivo involved restoring the dysfunctional bone morphogenetic protein receptor-2 (BMPR2) axis observed in PAH. This effect was only observed in female mice. In addition, chronic hypoxia induced- PH in male and female mice was unaffected by GPER deletion. Expression of GPER between female non-PAH controls and PAH patients was unchanged. In isolated human PASMCs estrogen induced proliferation was inhibited by MPP, but not PHTPP or G15, an ERβ and GPER antagonist, respectively. The ERα agonist, PPT stimulated proliferation of human PASMCs. Both estrogen and PPT induced proliferation was dependent on downstream PI3K/Akt and ERK MAPK activity. In males, testosterone deprivation by surgical castration had no effect on chronic-hypoxia induced PH. RVSP, RVH and pulmonary vascular remodelling were unchanged in hypoxic castrated mice relative to sham controls. Testosterone levels, assessed by enzyme linked immunosorbent assay (ELISA) demonstrated no effects of hypoxia on plasma testosterone levels. Testosterone levels were approximately halved by castration. qRT-PCR analysis showed that in mouse lung there were also no difference in expression of the androgen receptor (AR) and 5α-reducatse, the testosterone metabolising enzyme. Testosterone had no effect on proliferation of human PASMCs, although its primary metabolite, dihydrotestosterone (DHT), stimulated proliferation in a dose-dependent manner. In summary of these findings, we have identified an ERα-dependent mechanism of PAH in females, but not in males. ERα is noticeably increased in female human PASMCs from PAH patients compared to male PAH patients. Additionally, ERα activation in female human PASMCs leads to proliferation driven by PI3K/Akt and ERK MAPK activation. Treatment with an ERα antagonist attenuated the development of chronic hypoxia induced-PH in females but not males, and reversed PH in SERT+ female mice. We demonstrate that the mechanism attributed to the beneficial effect of MPP in vivo involved restoration of the dysfunctional BMPR2 signalling axis. Our results suggest that increased ERα expression may drive PAH development in females. Furthermore, we demonstrate that ERα does not play a key role in the development of hypoxia induced-PH in male mice. In addition we conclude that testosterone does not contribute to chronic hypoxic-PH observed in males. We suggest that altered local synthesis and metabolism in the lung and right ventricle may however, facilitate progression of established PAH in males and worsening survival rates. Overall, our results provide evidence for ERα in PAH development and implicate targeting ERs as a novel therapeutic target in PAH treatment.

Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

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The natural angiogenic response of the vasculature to cardiovascular disease has been shown, at least in part, to involve circulating endothelial progenitor cells (EPCs). However, the native response is often insufficient to restore vascularity without additional intervention. In this study the angiogenic activity of EPCs, demonstrated by in vitro tubule formation, confirmed the suggested potential of EPCs to be used therapeutically. However, as EPCs are found in limited circulating numbers, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were also investigated as sources of donor EPCs for transplantation. Here ESCs, but not iPSCs, were shown to generate cells with a genetic and proteomic profile, as well as an angiogenic potential, identical to natural EPCs. Using an in vivo mouse model of hindlimb ischemia, this investigation illustrated the preferential binding of transplanted EPCs at sites of angiogenic stimulation, and revealed the importance of platelets in the recruitment of circulating EPCs. In particular, using in vitro aggregation and flow-based adhesion assays, the adhesion molecule P-selectin was shown to play a significant role in this recruitment mechanism. In conclusion, this study has demonstrated that EPC transplantation has abundant potential for development into a viable and efficiacious therapeutic angiogenic treatment.

Tendon and ligament damage causes extreme pain and decreased joint functionality. Current repair methods cannot restore original joint biomechanics nor promote regeneration of native tissue. Recent advances in tendon and ligament repair have involved engineering tissue using cell-seeded scaffolds. Self-aligned cellular structures, similar to those in ligaments and tendons, have been successfully formed, albeit with weak attachment between construct and bone. Calcium phosphates form an intimate bond with both soft and hard tissues and have successfully been used in tissue engineering bone, whilst hydrogels have often been used as cellular scaffolds. This thesis explores agarose, gelatin, carrageenan and fibrin hydrogels as potential soft tissue scaffolds. Fibrin gel exhibited high cellular compatibility with highest metabolic activity on day 14. Although the cellular gel contracted significantly, it was found that the dry weight remained stable in both the acellular and cellular forms. 3D powder printed calcium phosphate scaffolds remained structurally stable after immersion in cell culture media with immersion in protein-rich sera promoting tenocyte attachment. Bracket designs were developed to enhance grip of the cell-seeded fibrin. Ligament constructs were selfsupporting and exhibited structural characteristics similar to native connective tissue. Tenocyte density peaked on day 14, with added L-proline and ascorbic acid inducing a constant level of glycosaminoglycans and 7.4 ± 1.5 % w/w collagen. This research may significantly enhance the clinical application of tissue engineered ligaments and tendons.