Chandra, Abhishek

Abstract Cellular senescence has been implicated in the pathophysiology of radiotherapy-associated bone loss. Based on our previous work, clearance of senescent cells using genetic and pharmacological tools alleviates the anomalies associated with radiation-associated bone deterioration. The pro-inflammatory senescence associated secretome referred to as senescence associated secretory phenotype (SASP), is a hallmark of cellular senescence. The modulation of SASP by senomorphic drugs, potentially can suppress the pro-inflammatory secretome of senescent cells, irrespective of the underlying senescence mechanism. In this study we tested a senomorphic drug, ruxolitinib, a Janus kinase inhibitor (JAKi), during acute and chronic radiotherapy related effects on the bone. Our clinical data indicate an early increase in several pro-inflammatory SASP proteins following radiotherapy of spinal metastasis in prostate cancer patients. Longitudinal assessment of SASP-related genes confirmed this acute elevation in several SASP markers in systemic circulation following irradiation of male mouse femurs. In studies done in male mice, following three preclinical radiotherapy regimens of 30Gy (5 x 6Gy), 60Gy (5 x 12Gy) and a single dose of 24Gy, suppression of SASP by JAKi was efficacious in suppressing radiation-induced bone damage. In comparison and as shown before, the senolytic combination of D+Q was also able to alleviate radiation-associated bone loss. Early and intermittent suppression of SASP using JAK inhibitors in male mice alleviated chronic bone deterioration, diminished telomere dysfunction, lowered senescence and SASP marker expression and reduced bone-marrow adiposity. Overall, our study shows that early targeting of SASP proteins could be a potential therapeutic to prevent radiotherapy-related chronic bone loss and risk of fractures.

Abstract Endothelial specialization is increasingly recognized as a fundamental regulator of tissue homeostasis, yet the cellular diversity of the skeletal vasculature remains incompletely resolved. Here, we integrate large-scale single-cell transcriptomics, cross-tissue comparisons, and imaging to comprehensively define endothelial heterogeneity across the skeleton. Our analyses demonstrate that the proposed post- arterial “type R” endothelial population is not a distinct endothelial subtype but instead comprises canonical venous endothelial cells and fatty acid-handling endothelial state. RNA velocity supports a venous continuum, while the proposed type R markers FMO2, and AQP7 lack both endothelial and skeletal specificity. The fatty acid-handling endothelial state, characterized by Lpl and Cd36 is conserved across multiple skeletal sites and non-skeletal tissues, indicating a general endothelial metabolic programme. Within bone, this endothelial state expands following high-fat diet and is suppressed during injury. Together, these findings redefine skeletal endothelial heterogeneity and establish the proposed type R population as part of a venous continuum.

SUMMARY This protocol describes a standardized and reproducible minimally invasive approach for establishing mouse models of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and osteoradionecrosis of the jaw (ORNJ). The method combines a unified low-trauma oral surgical procedure with disease-specific injury induction strategies to generate robust and clinically relevant models of jaw osteonecrosis. For BRONJ, systemic zoledronic acid administration is coupled with mandibular first molar extraction using tape-assisted mouth opening and customized bent micro-forceps, minimizing soft tissue damage and reducing procedural variability. For ORNJ, a customized lead-shielding platform enables precise, noninvasive mandible-targeted irradiation, producing reproducible bone injury while limiting off-target radiation exposure. Together, these complementary models provide a consistent and minimally invasive framework for investigating jaw osteonecrosis arising from distinct etiologies. The protocol supports comprehensive downstream analyses, including micro-computed tomography, histology, and immunofluorescence, and facilitates mechanistic studies of disease pathogenesis, bone regeneration, and therapeutic intervention.

Background/Objectives: Heterotopic ossification (HO), the aberrant formation of bone within soft tissues, arises either from rare genetic mutations or more commonly from traumatic insults. It is a major cause of morbidity not only in individuals harboring causative mutations, but also in those undergoing musculoskeletal surgery or trauma and in soldiers sustaining blast or burn injuries. Bone morphogenetic protein (BMP) signaling is a central driver of both hereditary and acquired forms of HO. Primary cilia are nonmotile, antenna-like organelles that extend from the cell surface and serve as crucial sensory and signaling hubs by concentrating key pathway components within a confined volume at the ciliary tip. However, their functional role in the pathogenesis of traumatic HO remains poorly understood. Methods: We investigate the role of primary cilia in traumatic HO using a genetically modified mouse model and cellular model. Results: We demonstrate that BMP signaling is attenuated when primary cilia function is disrupted. Both ciliation frequency and ciliary length were reduced in Scleraxis-CreERT2; Intraflagellar transport 88 floxed/floxed (Scx-CreERT2;Ift88fl/fl) tenocytes. Deletion of Ift88 effectively suppressed pathological BMP signaling and inhibited HO formation. Conclusions: These findings establish that functional primary cilia are required for traumatic HO development and highlight ciliary regulation as a potential therapeutic avenue for preventing or mitigating post-traumatic HO.

Abstract Cellular senescence has been implicated in the pathophysiology of radiotherapy-related bone loss. Based on our previous work, clearance of senescent cells using genetic and pharmacological tools alleviates the anomalies associated with radiation-associated bone deterioration. The pro-inflammatory senescence associated secretome referred to as senescence associated secretory phenotype (SASP), is a hallmark of cellular senescence. The modulation of SASP by senomorphic drugs, potentially can suppress the pro-inflammatory secretome of senescent cells, irrespective of the underlying senescence mechanism. In this study we tested a senomorphic drug, ruxolitinib, a Janus kinase inhibitor (JAKi), during acute and chronic radiotherapy related effects on the bone. Our clinical data indicate an early increase in several pro-inflammatory SASP proteins following radiotherapy of spinal metastasis in prostate cancer patients. Longitudinal assessment of SASP-related genes confirmed this acute elevation in several SASP markers in systemic circulation following irradiation of mouse femurs. In a proof-of-concept study, following two preclinical radiotherapy regimens of cumulative doses of 30Gy (5 x 6Gy) and 60Gy (5 x 12Gy), a senomorphic approach of JAKi treatment was more effective in alleviating radiation-related bone loss compared to the senolytic cocktail of D+Q. Early and intermittent suppression of SASP using JAK inhibitors alleviated chronic bone deterioration, diminished telomere dysfunction, lowered senescence and SASP marker expression, reduced bone-marrow adiposity, and mitigated radiation related lymphatic impairment. Overall, our study shows that early targeting of SASP proteins could be a potential therapeutic to prevent radiotherapy-related chronic bone loss and risk of fractures. Lay Summary Radiotherapy as part of cancer treatment is correlated with an acute increase in senescent cells. Here we show that the pro-inflammatory senescence associated secretory phenotype (SASP) becomes much more prominent soon after radiotherapy in both patients and mice. Suppression of the SASP using a Janus kinase inhibitor, ruxolitinib, reduced the overall senescent cell burden, improved bone architecture by promoting bone formation, reduced bone marrow adiposity, and mitigated radiation-induced lymphatic impairment. Overall, these findings suggest that early inhibition of the SASP may help mitigate several bone anomalies and prevent long-term bone loss and fractures after radiotherapy. Graphical Abstract

Abstract Cellular senescence is a key mechanism of skeletal aging in both physiological and accelerated conditions, such as radiotherapy. This study aimed to identify common differentially regulated microRNAs (miRs) across these contexts. We performed miR sequencing on three models: femurs from young (5-month-old) versus old (24-month-old) mice; focally radiated versus non-radiated femurs; and osteocytes from young versus old mice. Osteocytes were included in the comparison, as they have the longest lifespan in the mineralized bone matrix and they form 90-95% of all mesenchymal bone cell types. Among the three groups, miR-135a-5p and miR-671-5p were the common (i.e., shared) miRs that were downregulated, and miR-183-5p, a miR that regulates the WNT pathway, was the only shared upregulated miR, while miR-155-5p, a miR that regulates the Senescence-Associated Secretory Phenotype (SASP), was elevated in two conditions. The WNT-pathway has been positively associated with bone health and Sclerostin, a WNT-pathway inhibitor produced and secreted by osteocytes, has been implicated in accelerated skeletal deterioration following radiation. Thus, we used a neutralizing antibody to Sclerostin (Scl-Ab), to assess genes related to the WNT pathway and senescence, which are regulated by miR-183-5p and miR-155-5p, respectively. We further performed miR sequencing in radiated bones from mice treated with Scl-Ab and identified miR-133a-3p, a key miR that inhibits bone metabolism and function, which is upregulated in accelerated skeletal aging (i.e., focal radiation) downregulated by Scl-Ab. Overall, our study identifies potential regulatory gene pathways that modulate skeletal aging in the presence and absence of a WNT activator, Scl-Ab.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21 + cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21 + cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21 + cell clearance did not alter bone loss due to aging; conversely, p16 + cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21 + neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21 + OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell–like OCH subset that was p21 + and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21 + versus p16 + senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Due to modern medical advancements, greater proportions of the population will continue to age with longer life spans. Increased life span, however, does not always correlate with improved health span, and may result in an increase in aging-related diseases and disorders. These diseases are often attributed to cellular senescence, in which cells become disengaged from the cell cycle and inert to cell death. These cells are characterized by a proinflammatory secretome. The proinflammatory senescence-associated secretory phenotype, although part of a natural function intended to prevent further DNA damage, creates a microenvironment suited to tumor progression. This microenvironment is most evident in the gastrointestinal tract (GI), where a combination of bacterial infections, senescent cells, and inflammatory proteins can lead to oncogenesis. Thus, it is important to find potential senescence biomarkers as targets of novel therapies for GI diseases and disorders including cancers. However, finding therapeutic targets in the GI microenvironment to reduce the risk of GI tumor onset may also be of value. This review summarizes the effects of cellular senescence on GI aging, inflammation, and cancers, and aims to improve our understanding of these processes with a goal of enhancing future therapy.

Wnt signaling involves multiple pathways that contribute to organ development, cell fate, inflammation, and normal stem cell renewal and maintenance. Although the homeostasis of stem cells in the gastrointestinal (GI) tract highly depends on the Wnt signaling pathway, this regulation is impaired in cancers and in aging. Overactive (uncontrolled) Wnt signaling can induce GI epithelial cancers such as colon and gastric cancer. Overactive Wnt signaling can also contribute to the initiation and progression of gastrointestinal stromal tumor, which is the most common human sarcoma occurring in the walls of the digestive organs, mainly the stomach and small intestine. Wnt expression is positively associated not only with the progression of oncogenesis but also with resistance to chemotherapy and radiotherapy. Of note, recent reports show that decreased Wnt signaling is related to intestinal stem cell aging and that overactivated Wnt signaling leads to gastric pacemaker stem cell aging in tunica muscularis. These findings indicate that Wnt signaling has different crucial aspects of cell fate determination with age in GI tunica mucosa and muscularis. In this review, we summarize the most recent advances in our understanding of Wnt signaling pathways and their role in regulating key aspects during development, carcinogenesis, inflammation, and aging, with the ultimate goal of identifying novel therapies.

Abstract Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16 Ink4a or p21 Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21 ‐ ATTAC mouse with the p21 Cip1 promoter driving a “suicide” transgene encoding an inducible caspase‐8 which, upon induction, selectively kills p21 Cip1 ‐expressing senescent cells. Next, we used the p21 ‐ ATTAC mouse and the established p16 ‐ INK ‐ ATTAC mouse to directly compare the contributions of p21 Cip1 versus p16 Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence—specifically, radiation‐induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation‐induced p21 Cip1 ‐ or p16 Ink4a ‐driven transcripts following senescent cell clearance in both models. However, only clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells prevented both radiation‐induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells also reduced several of the radiation‐induced pro‐inflammatory senescence‐associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation‐induced osteoporosis is driven predominantly by p21 Cip1 ‐ rather than p16 Ink4a ‐mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence‐associated conditions, including aging across tissues.