ABSTRACT
Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.
Subject(s)
Osteomyelitis/pathology , Penicillin-Binding Proteins/metabolism , Staphylococcal Infections/complications , Staphylococcus aureus/isolation & purification , Animals , Anti-Bacterial Agents/pharmacology , Female , Mice , Mice, Inbred BALB C , Osteomyelitis/drug therapy , Osteomyelitis/metabolism , Osteomyelitis/microbiology , Penicillin-Binding Proteins/genetics , Staphylococcal Infections/microbiology , Vancomycin/pharmacologyABSTRACT
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.
Subject(s)
Abnormalities, Multiple/genetics , Autoantigens/physiology , Colon/abnormalities , Cytoskeletal Proteins/physiology , Intestinal Pseudo-Obstruction/genetics , Muscle Proteins/physiology , Urinary Bladder/abnormalities , Animals , Autoantigens/genetics , Autoantigens/metabolism , Codon, Nonsense , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Female , Humans , Infant, Newborn , Mice , Muscle Contraction/genetics , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Smooth/physiologyABSTRACT
BACKGROUND: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. METHODS: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. RESULTS: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. CONCLUSIONS: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.
Subject(s)
Heart Failure/metabolism , Hypertrophy, Left Ventricular/metabolism , Mechanotransduction, Cellular , Myocytes, Cardiac/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Aged , Animals , Animals, Newborn , COS Cells , Case-Control Studies , Chlorocebus aethiops , Connexin 43/genetics , Connexin 43/metabolism , Female , Gene Expression Regulation , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Hypertrophy, Left Ventricular/genetics , Hypertrophy, Left Ventricular/pathology , Hypertrophy, Left Ventricular/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Middle Aged , Myocytes, Cardiac/ultrastructure , NIH 3T3 Cells , Single Molecule Imaging , Trans-Activators/deficiency , Trans-Activators/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Ventricular Function, Left , Ventricular RemodelingABSTRACT
BACKGROUND: The size of secondary calciprotein particles (CPP2) and the speed of transformation (T50) from primary calciprotein particles (CPP1) to CPP2 in serum may be associated with vascular calcification (VC) in patients with chronic kidney disease (CKD). METHODS: We developed a high throughput, microplate-based assay using dynamic light scattering (DLS) to measure the transformation of CPP1 to CPP2, hydrodynamic radius (Rh) of CPP1 and CPP2, T50 and aggregation of CPP2. We used this DLS assay to test the hypothesis that a large Rh of CPP2 and/or a fast T50 are associated with VC in 45 participants with CKD Stages 4-5 (22 without VC and 23 with VC) and 17 healthy volunteers (HV). VC was defined as a Kauppila score >6 or an Adragao score ≥3. RESULTS: CKD participants with VC had larger cumulants Rh of CPP2 {370 nm [interquartile range (IQR) 272-566]} compared with CKD participants without VC [212 nm (IQR 169-315)] and compared with HV [168 nm (IQR 145-352), P < 0.01 for each]. More CPP2 were in aggregates in CKD participants with VC than those without VC (70% versus 36%). The odds of having VC increased by 9% with every 10 nm increase in the Rh of CPP2, after adjusting for age, diabetes, serum calcium and phosphate [odds ratio 1.09, 95% confidence interval (CI) 1.03, 1.16, P = 0.005]. The area under the receiver operating characteristic curve for VC of CPP2 size was 0.75 (95% CI 0.60, 0.90). T50 was similar in CKD participants with and without VC, although both groups had a lower T50 than HV. CONCLUSIONS: Rh of CPP2, but not T50, is independently associated with VC in patients with CKD Stages 4-5.
Subject(s)
Calcium/blood , Phosphates/blood , Photometry/methods , Renal Insufficiency, Chronic/blood , Vascular Calcification/blood , Adult , Cross-Sectional Studies , Diabetes Mellitus , Female , Glomerular Filtration Rate , Humans , Hydrodynamics , Light , Male , Middle Aged , Odds Ratio , Regression Analysis , Renal Insufficiency, Chronic/complications , Scattering, Radiation , Vascular Calcification/complications , Young AdultABSTRACT
Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1⯵m non-motile cocci, S. aureus deforms smaller than 0.5⯵m in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.
Subject(s)
Osteocytes/microbiology , Osteomyelitis/genetics , Staphylococcal Infections/genetics , Staphylococcus aureus/pathogenicity , Animals , Cortical Bone/microbiology , Cortical Bone/pathology , Humans , Mice , Osteocytes/pathology , Osteomyelitis/microbiology , Osteomyelitis/pathology , Quorum Sensing/genetics , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/geneticsABSTRACT
An important pool of cardiovascular progenitor cells arises from the epicardium, a single layer of mesothelium lining the heart. Epicardium-derived progenitor cell (EPDC) formation requires epithelial-to-mesenchymal transition (EMT) and the subsequent migration of these cells into the sub-epicardial space. Although some of the physiological signals that promote EMT are understood, the functional mediators of EPDC motility and differentiation are not known. Here, we identify a novel regulatory mechanism of EPDC mobilization. Myocardin-related transcription factor (MRTF)-A and MRTF-B (MKL1 and MKL2, respectively) are enriched in the perinuclear space of epicardial cells during development. Transforming growth factor (TGF)-ß signaling and disassembly of cell contacts leads to nuclear accumulation of MRTFs and the activation of the motile gene expression program. Conditional ablation of Mrtfa and Mrtfb specifically in the epicardium disrupts cell migration and leads to sub-epicardial hemorrhage, partially stemming from the depletion of coronary pericytes. Using lineage-tracing analyses, we demonstrate that sub-epicardial pericytes arise from EPDCs in a process that requires the MRTF-dependent motile gene expression program. These findings provide novel mechanisms linking EPDC motility and differentiation, shed light on the transcriptional control of coronary microvascular maturation and suggest novel therapeutic strategies to manipulate epicardium-derived progenitor cells for cardiac repair.
Subject(s)
Cell Movement , Coronary Vessels/growth & development , Pericardium/cytology , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , COS Cells , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chlorocebus aethiops , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Pericardium/metabolism , Pericardium/ultrastructure , Pericytes/cytology , Pericytes/drug effects , Serum Response Factor/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transforming Growth Factor beta1/pharmacologyABSTRACT
DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538-2549, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Bone Marrow Cells/metabolism , Calcium Signaling , Cell Differentiation , Membrane Proteins/metabolism , NFATC Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Osteoclasts/metabolism , Osteogenesis , Osteopetrosis/metabolism , Active Transport, Cell Nucleus , Animals , Bone Marrow Cells/pathology , Cell Fusion , Cell Movement , Cell Shape , Cells, Cultured , Genetic Predisposition to Disease , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice, Knockout , Mutation , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Osteoclasts/pathology , Osteolysis/metabolism , Osteolysis/pathology , Osteolysis/physiopathology , Osteopetrosis/genetics , Osteopetrosis/pathology , Osteopetrosis/physiopathology , Phenotype , Protein Interaction Domains and Motifs , Time Factors , TransfectionABSTRACT
INTRODUCTION: Erythropoietin (EPO) has been identified as a neuroregenerative agent. We hypothesize that it may accelerate recovery after crush injury and may vary with crush severity. METHODS: Mice were randomized to mild, moderate, or severe crush of the sciatic nerve and were treated with EPO or vehicle control after injury. The sciatic function index (SFI) was monitored over the first week. Microstructural changes were analyzed by immunofluorescence for neurofilament (NF) and myelin (P0 ), and electron microscopy was used to assess ultrastructural changes. RESULTS: In moderate crush injuries, EPO significantly improved SFI at 7 days post-injury, an effect not observed with other severity levels. Increases in the ratio of P0 to NF were observed after EPO treatment in moderate crush injuries. Electron microscopy demonstrated endothelial cell hypertrophy in the EPO group. CONCLUSIONS: EPO accelerates recovery in moderately crushed nerves, which may be through effects on myelination and vascularization. Injury severity may influence the efficacy of EPO. Muscle Nerve 56: 143-151, 2017.
Subject(s)
Erythropoietin/therapeutic use , Recovery of Function/drug effects , Sciatic Neuropathy/drug therapy , Sciatic Neuropathy/physiopathology , Analysis of Variance , Animals , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Intermediate Filaments/metabolism , Intermediate Filaments/pathology , Mice , Mice, Inbred C57BL , Microscopy, Electron , Myelin P0 Protein/metabolism , Sciatic Nerve/drug effects , Sciatic Nerve/pathology , Sciatic Nerve/ultrastructure , Severity of Illness IndexABSTRACT
BACKGROUND: Several acetabular components utilizing novel ultraporous metal substrates have been introduced over the past decade. Collectively by design, they have a lower modulus of elasticity to reduce stress shielding, a higher coefficient of friction to enhance interference fit, and ultraporous surfaces to enhance osseointegration. However, little literature exists regarding their clinical performance. METHODS: This study compared the clinical and radiographic results of 109 hips in 95 patients using a Tritanium primary cup (Stryker, Mahwah, NJ) to an age, body mass index, and gender-matched cohort of 100 patients that received a contemporary cup (Stryker Trident PSL HA). RESULTS: At an average 4.24 + 1.49 years, implant survivorship of the Tritanium primary cup was 98.2%, with 2 cups revised for failure of osseointegration. One-year radiographs revealed radiolucent and radiosclerotic lines in 2 or more DeLee zones in 30.3% of cups and 3 zone involvement in 8.2%. These proportions increased (40.0% and 17.1%, respectively) at minimum 5-year follow-up. A comparison of 1 year and last follow-up radiographs revealed progression in 13.8%. Tritanium primary components with radiolucency in 2 or more zones exhibited significantly lower HHS at 2 years compared to all Trident peripheral self-locking (PSL) components (P < .0001) and Tritanium primary components with 1 zone or no radiolucency (P = .026). Scanning electron microscopy of a retrieved cup revealed local inflammatory reaction and no evidence of osseointegration. CONCLUSION: Despite adequate implant survivorship, over one third of Tritanium primary cups had 2 or more zone radiolucency at minimum 5-year follow-up with associated lower Harris hip scores.
Subject(s)
Arthroplasty, Replacement, Hip/instrumentation , Hip Prosthesis/statistics & numerical data , Titanium , Acetabulum/surgery , Aged , Female , Follow-Up Studies , Friction , Hip Joint/diagnostic imaging , Humans , Male , Microscopy, Electron, Scanning , Middle Aged , Osseointegration , RadiographyABSTRACT
Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.
Subject(s)
Glycolysis , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Osteosarcoma/metabolism , Oxygen Consumption , Animals , Cell Line, Tumor , Humans , Mice , Mitochondria/ultrastructure , Mitochondrial Membranes/ultrastructure , Osteosarcoma/ultrastructure , PermeabilityABSTRACT
Basal cell carcinoma (BCC) is the most common skin cancer. Skin cancers may present either as a non-invasive tumor or an invasive malignancy. The terminology of carcinoma in situ is used when the tumor is either just limited to epidermis or not present as single cells or nests in the dermis. However, currently the terminology superficial BCC is inappropriately used instead of BCC in situ when the skin cancer is limited to epidermis. In this study we compare the pathologic changes of superficial, nodular, and infiltrative BCCs using electron microscopy to identify the ultrastructural characteristics and validate the previously proposed terminology. Three cases of BCC (superficial BCC, nodular BCC, and infiltrative BCC) diagnosed by dermatopathologists at our institute were selected for review. Paraffin block tissues from these cases were sent for electron microscopy studies which demonstrated disruption of basal lamina in both nodular and infiltrative type of BCC, while it remains intact in BCC superficial type after extensive examination. Therefore, similar to other in situ skin cancers, there is no invasion of the neoplasm in superficial BCC into the dermis. Hence, the older term superficial BCC should be appropriately replaced with the newer terminology BCC in situ.
ABSTRACT
Cutibacterium acnes, part of normal skin flora, is increasingly recognized as an opportunistic pathogen capable of causing chronic prosthetic joint infections (PJI) associated with total hip and knee arthroplasty. However, there is a paucity of literature examining the pathogenesis of C. acnes during PJI. To study this, we developed an implant-associated osteomyelitis murine model in which 8-10-week-old C57BL6 mice were subjected to transtibial implantation of titanium or stainless-steel L-shaped pins contaminated with C. acnes. Postsurgery, mice were killed on Days 14 and 28 for terminal assessments of (1) bacterial load in bone, implant, and internal organs (heart, spleen, kidney, and liver), (2) bone osteolysis (micro-CT), (3) abscess formation (histology), and (4) systematic electron microscopy (EM). In vitro scanning EM (SEM) confirmed that C. acnes can form biofilms on stainless-steel and titanium implants. In mice, C. acnes could persist for 28 days in the tibia. Also, we observed C. acnes dissemination to internal organs. C. acnes chronic osteomyelitis revealed markedly reduced bone osteolysis and abscess formation compared to Staphylococcus aureus infections. Importantly, transmission EM (TEM) investigation revealed the presence of C. acnes within canaliculi, demonstrating that C. acnes can invade the osteocyte lacuno-canalicular networks (OLCN) within bone. Our preliminary pilot study, for the first time, revealed that the OLCN in bone can be a reservoir for C. acnes and potentially provides a novel mechanism of why C. acnes chronic implant-associated bone infections are difficult to treat.
ABSTRACT
In the embryonic heart, the activation of the mitochondrial electron transport chain (ETC) coincides with the closure of the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (mPTP). However, it remains to be established whether the absence of CypD has a regulatory effect on mitochondria during cardiac development. Using a variety of assays to analyze cardiac tissue from wildtype and CypD knockout mice from embryonic day (E)9.5 to adult, we found that mitochondrial structure, function, and metabolism show distinct transitions. Deletion of CypD altered the timing of these transitions as the mPTP was closed at all ages, leading to coupled ETC activity in the early embryo, decreased citrate synthase activity, and an altered metabolome particularly after birth. Our results suggest that manipulating CypD activity may control myocyte proliferation and differentiation and could be a tool to increase ATP production and cardiac function in immature hearts.
ABSTRACT
Arachnoid granulations (AG) are poorly investigated. Historical reports suggest that they regulate brain volume by passively transporting cerebrospinal fluid (CSF) into dural venous sinuses. Here, we studied the microstructure of cerebral AG in humans with the aim of understanding their roles in physiology. We discovered marked variations in AG size, lobation, location, content, and degree of surface encapsulation. High-resolution microscopy shows that AG consist of outer capsule and inner stromal core regions. The fine and porous framework suggests uncharacterized functions of AG in mechanical CSF filtration. Moreover, internal cytokine and immune cell enrichment imply unexplored neuroimmune properties of these structures that localize to the brain-meningeal lymphatic interface. Dramatic age-associated changes in AG structure are additionally identified. This study depicts for the first time microscopic networks of internal channels that communicate with perisinus spaces, suggesting that AG subserve important functions as transarachnoidal flow passageways. These data raise new theories regarding glymphatic-lymphatic coupling and mechanisms of CSF antigen clearance, homeostasis, and diseases.
Subject(s)
Bone Marrow , Lymphatic Vessels , Humans , Arachnoid/ultrastructure , Dura Mater , Lymphatic SystemABSTRACT
Lymphatic muscle cell (LMC) contractility and coverage of collecting lymphatic vessels (CLVs) are integral to effective lymphatic drainage and tissue homeostasis. In fact, defects in lymphatic contractility have been identified in various conditions, including rheumatoid arthritis, inflammatory bowel disease, and obesity. However, the fundamental role of LMCs in these pathologic processes is limited, primarily due to the difficulty in directly investigating the enigmatic nature of this poorly characterized cell type. LMCs are a unique cell type that exhibit dual tonic and phasic contractility with hybrid structural features of both vascular smooth muscle cells (VSMCs) and cardiac myocytes. While advances have been made in recent years to better understand the biochemistry and function of LMCs, central questions regarding their origins, investiture into CLVs, and homeostasis remain unanswered. To summarize these discoveries, unexplained experimental results, and critical future directions, here we provide a focused review of current knowledge and open questions related to LMC progenitor cells, recruitment, maintenance, and regeneration. We also highlight the high-priority research goal of identifying LMC-specific genes towards genetic conditional- inducible in vivo gain and loss of function studies. While our interest in LMCs has been focused on understanding lymphatic dysfunction in an arthritic flare, these concepts are integral to the broader field of lymphatic biology, and have important potential for clinical translation through targeted therapeutics to control lymphatic contractility and drainage.
Subject(s)
Arthritis, Rheumatoid , Lymphatic Vessels , Humans , Lymphatic Vessels/metabolism , Lymphatic Vessels/pathology , Muscle Cells , RegenerationABSTRACT
Objective: Inflammatory-erosive arthritis is exacerbated by dysfunction of joint-draining popliteal lymphatic vessels (PLVs). Synovial mast cells are known to be pro-inflammatory in rheumatoid arthritis (RA). In other settings they have anti-inflammatory and tissue reparative effects. Herein, we elucidate the role of mast cells on PLV function and inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-tg) mice that exhibit defects in PLVs commensurate with disease progression. Methods: Whole mount immunofluorescent microscopy, toluidine blue stained histology, scanning electron microscopy, and in silico bioinformatics were performed to phenotype and quantify PLV mast cells. Ankle bone volumes were assessed by µCT, while corresponding histology quantified synovitis and osteoclasts. Near-infrared indocyanine green imaging measured lymphatic clearance as an outcome of PLV draining function. Effects of genetic MC depletion were assessed via comparison of 4.5-month-old WT, TNF-tg, MC deficient KitW-sh/W-sh (cKit-/-), and TNF-tg x cKit-/- mice. Pharmacological inhibition of mast cells was assessed by treating TNF-tg mice with placebo or cromolyn sodium (3.15mg/kg/day) for 3-weeks. Results: PLVs are surrounded by MCT+/MCPT1+/MCPT4+ mast cells whose numbers are increased 2.8-fold in TNF-tg mice. The percentage of peri-vascular degranulating mast cells was inversely correlated with ICG clearance. A population of MCT+/MCPT1-/MCPT4- mast cells were embedded within the PLV structure. In silico single-cell RNA-seq (scRNAseq) analyses identified a population of PLV-associated mast cells (marker genes: Mcpt4, Cma1, Cpa3, Tpsb2, Kit, Fcer1a & Gata2) with enhanced TGFß-related signaling that are phenotypically distinct from known MC subsets in the Mouse Cell Atlas. cKit-/- mice have greater lymphatic defects than TNF-tg mice with exacerbation of lymphatic dysfunction and inflammatory-erosive arthritis in TNF-tg x cKit-/- vs. TNF-Tg mice. Cromolyn sodium therapy stabilized PLV mast cells, increased TNF-induced bone loss, synovitis, and osteoclasts, and decreased ICG clearance. Conclusions: Mast cells are required for normal lymphatic function. Genetic ablation and pharmacological inhibition of mast cells exacerbates TNF-induced inflammatory-erosive arthritis with decreased lymphatic clearance. Together, these findings support an inflammatory role of activated/degranulated peri-PLV mast cells during arthritic progression, and a homeostatic role of intra-PLV mast cells, in which loss of the latter dominantly exacerbates arthritis secondary to defects in joint-draining lymphatics, warranting investigation into specific cellular mechanisms.
Subject(s)
Arthritis, Experimental , Lymphatic Vessels , Synovitis , Mice , Animals , Mice, Transgenic , Mast Cells/pathology , Cromolyn Sodium , Arthritis, Experimental/pathology , Lymphatic Vessels/pathology , Synovitis/pathologyABSTRACT
Mitochondria are critical for metabolic homeostasis of the liver, and their dysfunction is a major cause of liver diseases. Optic atrophy 1 (OPA1) is a mitochondrial fusion protein with a role in cristae shaping. Disruption of OPA1 causes mitochondrial dysfunction. However, the role of OPA1 in liver function is poorly understood. In this study, we delete OPA1 in the fully developed liver of male mice. Unexpectedly, OPA1 liver knockout (LKO) mice are healthy with unaffected mitochondrial respiration, despite disrupted cristae morphology. OPA1 LKO induces a stress response that establishes a new homeostatic state for sustained liver function. Our data show that OPA1 is required for proper complex V assembly and that OPA1 LKO protects the liver from drug toxicity. Mechanistically, OPA1 LKO decreases toxic drug metabolism and confers resistance to the mitochondrial permeability transition. This study demonstrates that OPA1 is dispensable in the liver, and that the mitohormesis induced by OPA1 LKO prevents liver injury and contributes to liver resiliency.
Subject(s)
Mitochondrial Dynamics , Mitochondrial Proteins , Male , Animals , Mice , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Mitochondria/metabolism , Liver/metabolismABSTRACT
Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Osteomyelitis , Staphylococcal Infections , Mice , Animals , Vancomycin/therapeutic use , Methicillin/therapeutic use , Anti-Bacterial Agents/pharmacology , Methicillin Resistance , Staphylococcal Infections/drug therapy , Osseointegration , Disease Models, Animal , Osteomyelitis/drug therapyABSTRACT
Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.
Subject(s)
Mitochondria/pathology , Mitochondrial Size , Osteosarcoma/pathology , Cell Line, Tumor , Cell Proliferation , Cell Respiration , DNA-Binding Proteins/metabolism , Electron Transport , Humans , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Neoplasm Invasiveness , Osteosarcoma/metabolism , Oxygen/metabolismABSTRACT
We report the first case of primary solid pseudopapillary tumor of the ovary with aggressive behavior and fatal outcome in a 45-year-old woman. The patient presented with weight loss, decrease of appetite, and abdominal bloating for the last several weeks. Computed tomography scan revealed an ovarian mass, omental caking, complex ascites, and 2 hepatic lesions. The pancreas was unremarkable. Grossly, the ovarian mass showed severe capsular adhesion, and the cut surface was cystic and solid. On histologic examination, the tumor was composed of diffuse solid pseudopapillary and pseudocystic patterns. The neoplastic cells were uniform and round with very dispersed chromatin. The cytoplasm was faintly pink. There was mild atypia, but the mitotic rate was as high as 62 per 50 high-power field, and the Ki-67 was elevated at 20%. The tumor exhibited severe necrosis. Numerous foci of lymphovascular invasion were also seen. The tumor cells were positive for cytokeratin (focal) and for ß-catenin (cytoplasmic and nuclear patterns). They were negative for chromogranin, synaptophysin, thyroglobulin, calcitonin, hepatocyte-paraffin 1, epithelial membrane antigen, calretinin, and α-inhibin. Electron microscopic study revealed nests of tumor cells with oval nuclei. The cytoplasm contained numerous pleomorphic mitochondria interspersed among short strands of rough endoplasmic reticulum. The tumor involved the fallopian tube, omentum, cul-de-sac, and abdominal wall. The pelvic washing was also positive for tumor cells. Despite chemotherapy, the patient's condition had worsened, and she died of her disease 8 months after the initial diagnosis. We discuss the differential diagnosis of this tumor and the hypothesis of its origin.