Zoledronic acid improves bone quality and muscle function in a high bone turnover state.

SUMMARY: Zoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGFß release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significance: TGFß is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGFß causes several bone disorders and skeletal muscle weakness. Reducing excess TGFß release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acid's benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Frequently Used Conceptual Frameworks and Design Principles for Extended Reality in Health Professions Education.

Health professions education (HPE) has witnessed a dramatic increase in the use of extended reality (XR), but there is limited evidence that conceptual frameworks are being effectively employed in the design and implementation of XR. This paper introduces commonly utilized conceptual frameworks that can support the integration of XR into the learning process and design principles that can be helpful for the development and evaluation of XR educational applications. Each framework and design principle is summarized briefly, followed by a description of its applicability to XR for HPE and an example of such application.

Implementing Rubric-Based Peer Review for Video Microlecture Design in Health Professions Education.

Efficient and effective instructional materials designed for asynchronous learning are increasingly important in health professions curricula. Video microlectures are an effective instructional method, but many faculty lack training in applying best-practice multimedia principles to development of their own recorded microlectures. Here we report a rubric designed for use in a peer-review process to evaluate and improve microlectures. The one-page rubric provides a framework for application of multimedia principles and cognitive load theory to microlecture design. Quality improvement of microlectures following redesign according to rubric elements is supported by increased student viewership, which coincided with higher rubric peer review scores.

Effect of Video Conferencing on Student Academic Performance: Evidence from Preclinical Summative Assessment Scores.

Anecdotal evidence suggests learners experience fatigue and burnout from multiple hours on virtual platforms. We compared summative exam performance data of second year preclinical medical students in a medical neuroscience course over consecutive years in which interactive synchronous activities occurred in-person (2019) or entirely online (2020). Exam items that assessed interactive, synchronously delivered content in 2020 had mean scores that were significantly lower than 2019. Interestingly, summative exam performance in the preceding course showed no appreciable difference. Taken together, our findings suggest that prolonged use of virtual platforms in preclinical medical education might negatively impact the efficacy of synchronous learning.

Therapeutic targeting of immune checkpoints with small molecule inhibitors.

Immune checkpoints are known to contribute to tumor progression by enhancing cancer's ability to evade the immune system and metastasize. Immunotherapies, including monoclonal antibodies, have been developed to target specific immunosuppressive molecules on the membranes of cancer cells and have proven revolutionary in the field of oncology. Recently, small molecule inhibitors (SMIs) have gained increased attention in cancer research with potential applications in immunotherapy. SMIs have desirable benefits over large-molecule inhibitors, such as monoclonal antibodies, including greater cell permeability, organ specificity, longer half-lives, cheaper production costs, and the possibility for oral administration. This paper will review the mechanisms by which noteworthy and novel immune checkpoints contribute to tumor progression, and how they may be targeted by SMIs and epigenetic modifiers to offer possible adjuvants to established therapeutic regimens. SMIs target immune checkpoints in several ways, such as blocking signaling between tumorigenic factors, building immune tolerance, and direct inhibition via epigenetic repression of immune inhibitory molecules. Further investigation into combination therapies utilizing SMIs and conventional cancer therapies will uncover new treatment options that may provide better patient outcomes across a range of cancers.

Dehydration: A Multidisciplinary Case-Based Discussion for First-Year Medical Students.

Introduction: As many medical school curricula shift to integrated learning of multiple basic science topics as well as clinical concepts, there is an increasing need for instructional materials that incorporate multiple topics yet are targeted to the knowledge basis of first-year medical students. This interactive case-based session for first-year medical students centers on the clinical presentation and initial evaluation of a patient experiencing dehydration after running a marathon in a high-altitude city. Methods: After completion of assigned out-of-class preparation, students followed the patient from a healthy state to moderate dehydration over the course of two 2-hour class sessions. Throughout discussion of the case, students answered questions requiring them to integrate elements of cell biology, biochemistry, physiology, and clinical reasoning with minimal faculty involvement. The learning activity was administered at University of Illinois College of Medicine campuses in both a small-group setting (10 students, one faculty facilitator) and a large-group format (55-90 students, multiple faculty facilitators). Following the activity, we assessed student perceptions of the design and implementation of the materials as well as effectiveness at meeting the learning goals. Results: Of 198 students who participated in the case discussions on dehydration, the majority rated the case positively, indicated by a rating of good or excellent. Discussion: This multidisciplinary case on dehydration can be used early in medical education to introduce students to clinical scenarios while learning fundamental science content. An integrated approach to medical content and versatility with regard to class size make this case a valuable teaching tool.

Osteocyte-Intrinsic TGF-ß Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling.

Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-ß) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-ß controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-ß signaling (TßRIIocy-/-), we show that TGF-ß regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility.

The Role of TGFß in Bone-Muscle Crosstalk.

PURPOSE OF REVIEW: The role of bone-derived factors in regulation of skeletal muscle function is an important emerging aspect of research into bone-muscle crosstalk. Implications for this area of research are far reaching and include understanding skeletal muscle weakness in cancer, osteoporosis, cachexia, rare diseases of bone, and aging. RECENT FINDINGS: Recent research shows that bone-derived factors can lead to changes in the skeletal muscle. These changes can either be anabolic or catabolic, and we focus this review on the role of TGFß in driving oxidative stress and skeletal muscle weakness in the setting of osteolytic cancer in the bone. The bone is a preferred site for breast cancer metastasis and leads to pathological bone loss. Osteolytic cancer in the bone leads to release of TGFß from the bone via osteoclast-mediated bone destruction. Our appreciation of crosstalk between the muscle and bone has recently expanded beyond mechanical force-driven events to encompass a variety of signaling factors originating in one tissue and communicating to the other. This review summarizes some previously known mediators of bone-to-muscle signaling and also recent work identifying a new role for bone-derived TGFß as a cause of skeletal muscle weakness in the setting of osteolytic cancer in the bone. Multiple points of potential therapeutic intervention are discussed.

Aromatase inhibitor-induced bone loss increases the progression of estrogen receptor-negative breast cancer in bone and exacerbates muscle weakness in vivo.

Aromatase inhibitors (AIs) cause muscle weakness, bone loss, and joint pain in up to half of cancer patients. Preclinical studies have demonstrated that increased osteoclastic bone resorption can impair muscle contractility and prime the bone microenvironment to accelerate metastatic growth. We hypothesized that AI-induced bone loss could increase breast cancer progression in bone and exacerbate muscle weakness associated with bone metastases. Female athymic nude mice underwent ovariectomy (OVX) or sham surgery and were treated with vehicle or AI (letrozole; Let). An OVX-Let group was then further treated with bisphosphonate (zoledronic acid; Zol). At week three, trabecular bone volume was measured and mice were inoculated with MDA-MB-231 cells into the cardiac ventricle and followed for progression of bone metastases. Five weeks after tumor cell inoculation, tumor-induced osteolytic lesion area was increased in OVX-Let mice and reduced in OVX-Let-Zol mice compared to sham-vehicle. Tumor burden in bone was increased in OVX-Let mice relative to sham-vehicle and OVX-Let-Zol mice. At the termination of the study, muscle-specific force of the extensor digitorum longus muscle was reduced in OVX-Let mice compared to sham-vehicle mice, however, the addition of Zol improved muscle function. In summary, AI treatment induced bone loss and skeletal muscle weakness, recapitulating effects observed in cancer patients. Prevention of AI-induced osteoclastic bone resorption using a bisphosphonate attenuated the development of breast cancer bone metastases and improved muscle function in mice. These findings highlight the bone microenvironment as a modulator of tumor growth locally and muscle function systemically.

Osteolytic Breast Cancer Causes Skeletal Muscle Weakness in an Immunocompetent Syngeneic Mouse Model.

Muscle weakness and cachexia are significant paraneoplastic syndromes of many advanced cancers. Osteolytic bone metastases are common in advanced breast cancer and are a major contributor to decreased survival, performance, and quality of life for patients. Pathologic fracture caused by osteolytic cancer in bone (OCIB) leads to a significant (32%) increased risk of death compared to patients without fracture. Since muscle weakness is linked to risk of falls which are a major cause of fracture, we have investigated skeletal muscle response to OCIB. Here, we show that a syngeneic mouse model of OCIB (4T1 mammary tumor cells) leads to cachexia and skeletal muscle weakness associated with oxidation of the ryanodine receptor and calcium (Ca2+) release channel (RyR1). Muscle atrophy follows known pathways via both myostatin signaling and expression of muscle-specific ubiquitin ligases, atrogin-1 and MuRF1. We have identified a mechanism for skeletal muscle weakness due to increased oxidative stress on RyR1 via NAPDH oxidases [NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4)]. In addition, SMAD3 phosphorylation is higher in muscle from tumor-bearing mice, a critical step in the intracellular signaling pathway that transmits TGFß signaling to the nucleus. This is the first time that skeletal muscle weakness has been described in a syngeneic model of OCIB and represents a unique model system in which to study cachexia and changes in skeletal muscle.

The P2Y2 nucleotide receptor is an inhibitor of vascular calcification.

BACKGROUND AND AIMS: Mutations in the 5'-nucleotidase ecto (NT5E) gene that encodes CD73, a nucleotidase that converts AMP to adenosine, are linked to arterial calcification. However, the role of purinergic receptor signaling in the pathology of intimal calcification is not well understood. In this study, we examined whether extracellular nucleotides acting via P2Y2 receptor (P2Y2R) modulate arterial intimal calcification, a condition highly correlated with cardiovascular morbidity. METHODS: Apolipoprotein E, P2Y2R double knockout mice (ApoE-/-P2Y2R-/-) were used to determine the effect of P2Y2R deficiency on vascular calcification in vivo. Vascular smooth muscle cells (VSMC) isolated from P2Y2R-/- mice grown in high phosphate medium were used to assess the role of P2Y2R in the conversion of VSMC into osteoblasts. Luciferase-reporter assays were used to assess the effect of P2Y2R on the transcriptional activity of Runx2. RESULTS: P2Y2R deficiency in ApoE-/- mice caused extensive intimal calcification despite a significant reduction in atherosclerosis and macrophage plaque content. The ectoenzyme apyrase that degrades nucleoside di- and triphosphates accelerated high phosphate-induced calcium deposition in cultured VSMC. Expression of P2Y2R inhibits calcification in vitro inhibited the osteoblastic trans-differentiation of VSMC. Mechanistically, expression of P2Y2R inhibited Runx2 transcriptional activation of an osteocalcin promoter driven luciferase reporter gene. CONCLUSIONS: This study reveals a role for vascular P2Y2R as an inhibitor of arterial intimal calcification and provides a new mechanistic insight into the regulation of the osteoblastic trans-differentiation of SMC through P2Y2R-mediated Runx2 antagonism. Given that calcification of atherosclerotic lesions is a significant clinical problem, activating P2Y2R may be an effective therapeutic approach for treatment or prevention of vascular calcification.

Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

RATIONALE: The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. OBJECTIVE: Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. METHODS AND RESULTS: Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. CONCLUSIONS: We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

Skeletal muscle Ca(2+) mishandling: Another effect of bone-to-muscle signaling.

Our appreciation of crosstalk between muscle and bone has recently expanded beyond mechanical force-driven events to encompass a variety of signaling factors originating in one tissue and communicating to the other. While the recent identification of new 'myokines' has shifted some focus to the role of muscle in this partnership, bone-derived factors and their effects on skeletal muscle should not be overlooked. This review summarizes some previously known mediators of bone-to-muscle signaling and also recent work identifying a new role for bone-derived TGF-ß as a cause of skeletal muscle weakness in the setting of cancer-induced bone destruction. Oxidation of the ryanodine receptor/calcium release channel (RyR1) in skeletal muscle occurs via a TGF-ß-Nox4-RyR1 axis and leads to calcium mishandling and decreased muscle function. Multiple points of potential therapeutic intervention were identified, from preventing the bone destruction to stabilizing the RYR1 calcium channel. This new data reinforces the concept that bone can be an important source of signaling factors in pathphysiological settings.

Up-regulation of glycolytic metabolism is required for HIF1α-driven bone formation.

The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation.

Radioactive in situ hybridization to detect gene expression in skeletal tissue sections.

In situ hybridization (ISH) using RNA probes is a valuable technique to characterize gene expression patterns in animal tissues. It provides valuable spatial information about gene expression. Compared to the nonradioactive alternatives,(35)S radioactive ISH generally provides higher sensitivity. Here, we describe the procedure for(35)S ISH on paraffin sections from the skeletal tissues of mouse embryos.

Osx-Cre targets multiple cell types besides osteoblast lineage in postnatal mice.

Osterix (Osx or Sp7) is a zinc-finger-family transcriptional factor essential for osteoblast differentiation in mammals. The Osx-Cre mouse line (also known as Osx1-GFP::Cre) expresses GFP::Cre fusion protein from a BAC transgene containing the Osx regulatory sequence. The mouse strain was initially characterized during embryogenesis, and found to target mainly osteoblast-lineage cells. Because the strain has been increasingly used in postnatal studies, it is important to evaluate its targeting specificity in mice after birth. By crossing the Osx-Cre mouse with the R26-mT/mG reporter line and analyzing the progenies at two months of age, we find that Osx-Cre targets not only osteoblasts, osteocytes and hypertrophic chondrocytes as expected, but also stromal cells, adipocytes and perivascular cells in the bone marrow. The targeting of adipocytes and perivascular cells appears to be specific to those residing within the bone marrow, as the same cell types elsewhere are not targeted. Beyond the skeleton, Osx-Cre also targets the olfactory glomerular cells, and a subset of the gastric and intestinal epithelium. Thus, potential contributions from the non-osteoblast-lineage cells should be considered when Osx-Cre is used to study gene functions in postnatal mice.

Tbx18 regulates development of the epicardium and coronary vessels.

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18(-/-) mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α(lacZ/+) activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18(-/-) hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.

Notch signaling and bone remodeling.

Notch signaling plays context-dependent roles in the development and maintenance of many cell types and tissues in mammals. In the skeleton, both osteoblasts and osteoclasts require Notch signaling for proper differentiation and function, and the specific roles of Notch are dependent on the differentiation status of the cell. The recent discovery of activating NOTCH2 mutations as the cause of Hajdu-Cheney syndrome has highlighted the significance of Notch signaling in human bone physiology.

Wnt1/ßcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair.

Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/ßcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial-mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/ßcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/ßcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury.

Vascular smooth muscle progenitor cells: building and repairing blood vessels.

Molecular pathways that control the specification, migration, and number of available smooth muscle progenitor cells play key roles in determining blood vessel size and structure, capacity for tissue repair, and progression of age-related disorders. Defects in these pathways produce malformations of developing blood vessels, depletion of smooth muscle progenitor cell pools for vessel wall maintenance and repair, and aberrant activation of alternative differentiation pathways in vascular disease. A better understanding of the molecular mechanisms that uniquely specify and maintain vascular smooth muscle cell precursors is essential if we are to use advances in stem and progenitor cell biology and somatic cell reprogramming for applications directed to the vessel wall.