Endometrioid Carcinomas of the Ovaries and Endometrium Involving Endocervical Polyps: Comprehensive Clinicopathological Analyses.

While synchronous ovarian and endometrial endometrioid carcinomas (ECs) have long been described in the literature, ovarian or endometrial EC involving concomitant endocervical polyp (ECP) has not yet been reported. This study aimed to investigate the histological types and prevalence of gynecological tumors co-existing with ECP and to comprehensively analyze the clinicopathological characteristics of ovarian and endometrial ECs involving ECPs. We searched for ECP cases associated with premalignant lesions or malignancies of the female genital tract occurring between March 2019 and February 2022. We then investigated the histological types and prevalence of gynecological tumors co-existing with ECP. In addition, we reviewed electronic medical records and pathology slides to collect the clinicopathological features of four patients with ovarian or endometrial EC involving ECP. We found 429 ECPs over the three-year study period. Of these, 68 (15.9%) were associated with premalignant or malignant lesions occurring in the uterine cervix, endometrium, and ovaries. Four of these cases, including two (0.5%) ovarian grade 3 ECs and two (0.5%) endometrial grade 1 ECs, involved ECPs. In the former cases (cases 1 and 2), ECs involving ECPs exhibited similar morphology and immunohistochemical staining results to those of advanced-stage ovarian EC. In the latter cases (cases 3 and 4), the histological and immunophenotypical features of EC involving ECP were identical to those of primary endometrial EC, despite the lack of tumor involvement in the myometrium, lower uterine segment, and cervical stroma as well as the absence of lymphovascular invasion and lymph node metastasis. In all cases, no evidence of benign endometriosis, endometrial hyperplasia without atypia, or atypical hyperplasia/endometrial intraepithelial neoplasm within ECP or the adjacent endocervical tissue was noted. Considering our results, the involvement of ECP by EC may have been caused by an implantation metastasis from the ovarian (cases 1 and 2) or endometrial (cases 3 and 4) EC. To the best of our knowledge, this is the first exploration of the synchronous occurrence of endometrial or ovarian EC and ECP involvement. Implantation metastasis via transtubal and trans-endometrial cavity migration may have been the pathogenic mechanism of ECP involvement.

A new murine model of Barth syndrome neutropenia links TAFAZZIN deficiency to increased ER stress-induced apoptosis.

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy, and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knockout (KO) animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN-KO hematopoietic stem cells. We also used the ER-Hoxb8 system (estrogen receptor fused to Hoxb8) of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN-KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress-mediated and non-ER stress-mediated triggers of apoptosis. Although the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some patients with Barth syndrome remains to be clarified, our studies and new model system set a foundation for further investigation.

Nusinersen by subcutaneous intrathecal catheter for symptomatic spinal muscular atrophy patients with complex spine anatomy.

INTRODUCTION/AIMS: Intrathecal administration of nusinersen is challenging in patients with spinal muscular atrophy (SMA) who have spine deformities or fusions. We prospectively studied the safety and efficacy of nusinersen administration via an indwelling subcutaneous intrathecal catheter (SIC) for SMA patients with advanced disease. METHODS: Seventeen participants commenced nusinersen therapy between 2.7 and 31.5 years of age and received 9 to 12 doses via SIC. Safety was assessed in all participants. A separate efficacy analysis comprised 11 nonambulatory, treatment-naive SMA patients (18.1 ± 6.8 years) with three SMN2 copies and complex spine anatomy. RESULTS: In the safety analysis, 14 treatment-related adverse events (AEs) occurred among 12 (71%) participants; all were related to the SIC and not nusinersen. Device-related AEs interfered with 2.5% of nusinersen doses. Four SICs (24%) required surgical revision due to mechanical malfunction with or without cerebrospinal fluid leak (n = 2), and one (6%) was removed due to Staphylococcus epidermidis meningitis. In the efficacy analysis, mean performance on the nine-hole peg test improved in dominant (15.9%, P = 0.012) and nondominant (19.0%, P = 0.008) hands and grip strength increased by 44.9% (P = 0.031). We observed no significant changes in motor scales, muscle force, pulmonary function, or SMA biomarkers. All participants in the efficacy cohort reported one or more subjective improvement(s) in endurance, purposeful hand use, arm strength, head control, and/or speech. DISCUSSION: For SMA patients with complex spine anatomy, the SIC allows for reliable outpatient administration of nusinersen that results in meaningful improvements in upper limb function, but introduces risks of technical malfunction and iatrogenic infection.

Implications of circulating neurofilaments for spinal muscular atrophy treatment early in life: A case series.

This longitudinal cohort study aimed to determine whether circulating neurofilaments (NFs) can monitor response to molecular therapies in newborns with spinal muscular atrophy (SMA; NCT02831296). We applied a mixed-effect model to examine differences in serum NF levels among healthy control infants (n = 13), untreated SMA infants (n = 68), and SMA infants who received the genetic therapies nusinersen and/or onasemnogene abeparvovec (n = 22). Increased NF levels were inversely associated with SMN2 copy number. SMA infants treated with either nusinersen or onasemnogene abeparvovec achieved important motor milestones not observed in the untreated cohort. NF levels declined more rapidly in the nusinersen cohort as compared with the untreated cohort. Unexpectedly, those receiving onasemnogene abeparvovec monotherapy showed a significant rise in NF levels regardless of SMN2 copy number. In contrast, symptomatic SMA infants who received nusinersen, followed by onasemnogene abeparvovec within a short interval after, did not show an elevation in NF levels. While NF cannot be used as the single marker to predict outcomes, the elevated NF levels observed with onasemnogene abeparvovec and its absence in infants treated first with nusinersen may indicate a protective effect of co-therapy during a critical period of vulnerability to acute denervation.

Caveolin-1 mediates soft scaffold-enhanced adipogenesis of human mesenchymal stem cells.

BACKGROUND: Human bone marrow-derived mesenchymal stem cells (hBMSCs) can differentiate into adipocytes upon stimulation and are considered an appropriate cell source for adipose tissue engineering. In addition to biochemical cues, the stiffness of a substrate that cells attach to has also been shown to affect hBMSC differentiation potential. Of note, most current studies are conducted on monolayer cultures which do not directly inform adipose tissue engineering, where 3-dimensional (3D) scaffolds are often used to create proper tissue architecture. In this study, we aim to examine the adipogenic differentiation of hBMSCs within soft or stiff scaffolds and investigate the molecular mechanism mediating the response of hBMSCs to substrate stiffness in 3D culture, specifically the involvement of the integral membrane protein, caveolin-1 (CAV1), known to regulate signaling in MSCs via compartmentalizing and concentrating signaling molecules. METHODS: By adjusting the photo-illumination time, photocrosslinkable gelatin scaffolds with the same polymer concentration but different stiffnesses were created. hBMSCs were seeded within soft and stiff scaffolds, and their response to adipogenic induction under different substrate mechanical conditions was characterized. The functional involvement of CAV1 was assessed by suppressing its expression level using CAV1-specific siRNA. RESULTS: The soft and stiff scaffolds used in this study had a compressive modulus of ~0.5 kPa and ~23.5 kPa, respectively. hBMSCs showed high viability in both scaffold types, but only spread out in the soft scaffolds. hBMSCs cultured in soft scaffolds displayed significantly higher adipogenesis, as revealed by histology, qRT-PCR, and immunostaining. Interestingly, a lower CAV1 level was observed in hBMSCs in the soft scaffolds, concomitantly accompanied by increased levels of Yes-associated protein (YAP) and decreased YAP phosphorylation, when compared to cells seeded in the stiff scaffolds. Interestingly, reducing CAV1 expression with siRNA was shown to further enhance hBMSC adipogenesis, which may function through activation of the YAP signaling pathway. CONCLUSIONS: Soft biomaterials support superior adipogenesis of encapsulated hBMSCs in 3D culture, which is partially mediated by the CAV1-YAP axis. Suppressing CAV1 expression levels represents a robust method in the promotion of hBMSC adipogenesis.

14-Year Changes in Plasma Neurofilament Levels Among Men: A Pilot Study.

Little is known about the feasibility of using long-term stored blood samples to measure neurofilament levels and about long-term changes in neurofilament levels among healthy individuals. We performed a pilot study among 26 adult men in preparation for a larger-scale study of the natural history of neurofilament levels. Median change over 14 years in pNf-H was 97.1 pg/mL (IQR: 5.0 to 242.0 pg/mL) and in Nf-L was 2.117 pg/mL (IQR: -2.691 to 3.393 pg/mL). We demonstrated the feasibility of measuring neurofilament concentrations in stored blood samples and found a trend between age and increases in Nf-L levels among adults.

Muscle injury promotes heterotopic ossification by stimulating local bone morphogenetic protein-7 production.

BACKGROUND: Heterotopic ossification (HO) is a pathological condition of abnormal bone formation in soft tissue, which causes pain and restricted range of motion in patients. There are two broad categories of HO, hereditary and acquired. Although different types of HO do not use identical mechanistic pathways of pathogenesis, muscle injury appears to be a unifying feature for all types of HO. However, little is known about the mechanisms by which muscle injury facilitates HO formation. OBJECTIVE AND METHOD: This study aimed to explore the cellular and molecular mechanisms linking muscle injury to HO by using cardiotoxin to induce muscle injury in a bone morphogenetic protein-2 (BMP-2)-induced HO mouse model. RESULTS: We found that muscle injury augmented HO formation and that this effect was correlated with BMP signalling activation and upregulation of BMP-7 expression at the early phase of HO progression. We further demonstrated that inhibition of BMP-7 activity in vitro suppressed the osteogenesis-promoting effect of conditioned medium derived from injured muscle tissue and in vivo reduced the volume of HO formation. We also showed that antiinflammatory drug treatment reduced the volume of HO with concomitant reduction in BMP-7 production. CONCLUSION: In summary, our study has identified BMP-7 as a key osteoinductive factor in injured muscle that facilitates HO formation. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our results provide a candidate mechanistic rationale for the use of antiinflammatory drugs in the prevention of HO.

Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect.

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106  cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.

Bone marrow mesenchymal stem cells: Aging and tissue engineering applications to enhance bone healing.

Bone has well documented natural healing capacity that normally is sufficient to repair fractures and other common injuries. However, the properties of bone change throughout life, and aging is accompanied by increased incidence of bone diseases and compromised fracture healing capacity, which necessitate effective therapies capable of enhancing bone regeneration. The therapeutic potential of adult mesenchymal stem cells (MSCs) for bone repair has been long proposed and examined. Actions of MSCs may include direct differentiation to become bone cells, attraction and recruitment of other cells, or creation of a regenerative environment via production of trophic growth factors. With systemic aging, MSCs also undergo functional decline, which has been well investigated in a number of recent studies. In this review, we first describe the changes in MSCs during aging and discuss how these alterations can affect bone regeneration. We next review current research findings on bone tissue engineering, which is considered a promising and viable therapeutic solution for structural and functional restoration of bone. In particular, the importance of MSCs and bioscaffolds is highlighted. Finally, potential approaches for the prevention of MSC aging and the rejuvenation of aged MSC are discussed.

Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells.

BACKGROUND: Adult mesenchymal stem cells (MSCs) are an important resource for tissue growth, repair, and regeneration. To utilize MSCs more effectively, a clear understanding of how they react to environmental cues is essential. Currently, relatively little is known about how the composition of the plasma membranes affects stem cell phenotype and properties. The presence of lipid molecules, including cholesterol in particular, in the plasma membrane plays a crucial role in regulating a variety of physiological processes in cells. In this study, we examined the effects of perturbations in cholesterol/caveolin-1 (CAV-1)/caveolae homeostasis on the membrane properties and adhesive characteristics of MSCs. Findings from this study will contribute to the understanding of how cholesterol/CAV-1/caveolae regulates aspects of the cell membrane important to cell adhesion, substrate sensing, and microenvironment interaction. METHODS: We generated five experimental MSC groups: 1) untreated MSCs; 2) cholesterol-depleted MSCs; 3) cholesterol-supplemented MSCs; 4) MSCs transfected with control, nonspecific small interfering (si)RNA; and 5) MSCs transfected with CAV-1 siRNA. Each cell group was analyzed for perturbation of cholesterol status and CAV-1 expression by performing Amplex Red cholesterol assay, filipin fluorescence staining, and real-time polymerase chain reaction (PCR). The membrane fluidity in the five experimental cell groups were measured using pyrene fluorescence probe staining followed by FACS analysis. Cell adhesion to collagen and fibronectin as well as cell surface integrin expression were examined. RESULTS: Cholesterol supplementation to MSCs increased membrane cholesterol, and resulted in decreased membrane fluidity and localization of elevated numbers of caveolae and CAV-1 to the cell membrane. These cells showed increased expression of α1, α4, and ß1 integrins, and exhibited higher adhesion rates to fibronectin and collagen. Conversely, knockdown of CAV-1 expression or cholesterol depletion on MSCs caused a parallel decrease in caveolae content and an increase in membrane fluidity due to decreased delivery of cholesterol to the cell membrane. Cells with depleted CAV-1 expression showed decreased cell surface integrin expression and slower adhesion to different substrates. CONCLUSIONS: Our results demonstrate that perturbations in cholesterol/CAV-1 levels significantly affect the membrane properties of MSCs. These findings suggest that modification of membrane cholesterol and/or CAV-1 and caveolae may be used to manipulate the biological activities of MSCs.

From embryonic development to human diseases: The functional role of caveolae/caveolin.

Caveolae, an almost ubiquitous, structural component of the plasma membrane, play a critical role in many functions essential for proper cell function, including membrane trafficking, signal transduction, extracellular matrix remodeling, and tissue regeneration. Three main types of caveolin proteins have been identified from caveolae since the discovery of caveolin-1 in the early 1990s. All three (Cav-1, Cav-2, and Cav-3) play crucial roles in mammalian physiology, and can effect pathogenesis in a wide range of human diseases. While many biological activities of caveolins have been uncovered since its discovery, their role and regulation in embryonic develop remain largely poorly understood, although there is increasing evidence that caveolins may be linked to lung and brain birth defects. Further investigations are clearly needed to decipher how caveolae/caveolins mediate cellular functions and activities of normal embryogenesis and how their perturbations contribute to developmental disorders.

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis.

INTRODUCTION: Stem cells are considered an important resource for tissue repair and regeneration. Their utilization in regenerative medicine will be aided by mechanistic insight into their responsiveness to external stimuli. It is likely that, similar to all other cells, an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts. The clustering of signaling molecules in these cholesterol-rich membrane microdomains can affect the activity, specificity, cross-talk and amplification of cell signaling. Membrane rafts fall into two broad categories, non-caveolar and caveolar, based on the absence or presence, respectively, of caveolin scaffolding proteins. We have recently demonstrated that caveolin-1 (Cav-1) expression increases during, and knockdown of Cav-1 expression enhances, osteogenic differentiation of human bone marrow derived mesenchymal stem cells (MSCs). The increase in Cav-1 expression observed during osteogenesis is likely a negative feedback mechanism. We hypothesize that focal adhesion signaling pathways such as PI3K/Akt signaling may be negatively regulated by Cav-1 during human MSC osteogenesis. METHODS: Human bone marrow MSCs were isolated from femoral heads obtained after total hip arthroplasty. MSCs were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition of supplements (ß-glycerophosphate, ascorbic acid, dexamethasone, and 1,25-dihydroxyvitamin D3). The activation of and requirement for PI3K/Akt signaling in MSC osteogenesis were assessed by immunoblotting for phosphorylated Akt, and treatment with the PI3K inhibitor LY294002 and Akt siRNA, respectively. The influences of Cav-1 and cholesterol membrane rafts on PI3K/Akt signaling were investigated by treatment with Cav-1 siRNA, methyl-ß-cyclodextrin, or cholesterol oxidase, followed by cellular sub-fractionation and/or immunoblotting for phosphorylated Akt. RESULTS: LY294002 and Akt siRNA inhibited MSC osteogenesis. Methyl-ß-cyclodextrin, which disrupts all membrane rafts, inhibited osteogenesis. Conversely, Cav-1 siRNA and cholesterol oxidase, which displaces Cav-1 from caveolae, enhanced Akt signaling induced by osteogenic supplements. In control cells, phosphorylated Akt began to accumulate in caveolae after 10 days of osteogenic differentiation. CONCLUSIONS: PI3K/Akt signaling is a key pathway required for human MSC osteogenesis, and it is likely that localization of active Akt in non-caveolar and caveolar membrane rafts positively and negatively contributes to osteogenesis, respectively.

Activation of non-myogenic mesenchymal stem cells during the disease progression in dystrophic dystrophin/utrophin knockout mice.

Ectopic calcification as well as fatty and fibrotic tissue accumulation occurs in skeletal muscle during the disease progression of Duchenne muscular dystrophy (DMD), a degenerative muscle disorder caused by mutations in the dystrophin gene. The cellular origin and the environmental cues responsible for this ectopic calcification, fatty and fibrotic infiltration during the disease progression, however, remain unknown. Based on a previously published preplate technique, we isolated two distinct populations of muscle-derived cells from skeletal muscle: (i) a rapidly adhering cell population, which is non-myogenic, Pax7(-) and express the mesenchymal stem cell (MSC) marker platelet-derived growth factor receptor alpha; hence, we termed this population of cells non-myogenic MSCs (nmMSCs); and (ii) a slowly adhering cell population which is Pax7(+) and highly myogenic, termed muscle progenitor cells (MPCs). Previously, we demonstrated that the rapid progression of skeletal muscle histopathologies in dystrophin/utrophin knockout (dys(-/-) utro(-/-) dKO) mice is closely associated with a rapid depletion of the MPC population pool. In the current study, we showed that in contrast to the MPCs, the nmMSCs become activated during the disease progression in dKO mice, displaying increased proliferation and differentiation potentials (adipogenesis, osteogenesis and fibrogenesis). We also found that after co-culturing the dKO-nmMSCs with dKO-MPCs, the myogenic differentiation potential of the dKO-MPCs was reduced. This effect was found to be potentially mediated by the secretion of secreted frizzled-related protein 1 by the dKO-nmMSCs. We therefore posit that the rapid occurrence of fibrosis, ectopic calcification and fat accumulation, in dKO mice, is not only attributable to the rapid depletion of the MPC pool, but is also the consequence of nmMSC activation. Results from this study suggest that approaches to alleviate muscle weakness and wasting in DMD patients should not only target the myogenic MPCs but should also attempt to prevent the activation of the nmMSCs.

Rapid depletion of muscle progenitor cells in dystrophic mdx/utrophin-/- mice.

Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3-5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients.