Unilateral Lung Removal in Combination with Monocrotaline or SU5416 in Rodents: A Reliable Model to Mimic the Pathology of the Human Pulmonary Hypertension.

Pulmonary hypertension (PH) is a chronic and progressive disorder characterized by elevated mean pulmonary arterial pressure, pulmonary vascular remodeling, and the development of concentric laminar intimal fibrosis with plexiform lesions. While rodent models have been developed to study PH, they have certain deficiencies and do not entirely replicate the human disease due to the heterogeneity of PH pathology. Therefore, combined models are necessary to study PH. Recent studies have shown that altered pulmonary blood flow is a significant trigger in the development of vascular remodeling and neointimal lesions. One of the most promising rodent models for increased pulmonary flow is the combination of unilateral left pneumonectomy with a "second hit" of monocrotaline (MCT) or SU5416. The removal of one lung in this model forces blood to circulate only in the other lung and induces increased and turbulent pulmonary blood flow. This increased vascular flow leads to progressive remodeling and occlusion of small pulmonary arteries. The second hit by MCT or SU5416 leads to endothelial cell dysfunction, resulting in severe PH and the development of plexiform arteriopathy.

Effects of Hormone Replacement Therapy on Women's Lung Health and Disease.

This review provides an overview of menopausal hormone therapy and pulmonary disease risk, with a focus on the effect of hormone replacement therapy (HRT) on pulmonary function and its relation to lung diseases. This summary is based on authors' knowledge in the field of HRT and supplemented by a PubMed search using the terms "menopause hormone therapy," "asthma", "lung cancer", "chronic obstructive pulmonary disease", "lung function", and "pulmonary hypertension". Available evidence indicates that there is limited research on the role of sex hormones in the susceptibility, severity, and progression of chronic respiratory diseases. However, some studies suggest that the hormonal changes that occur during the menopausal transition may have an impact on pulmonary function and respiratory diseases. Women are in need of convenient access to a safe and effective modality for personalized HRT based on an artificial intelligence (AI)-driven platform that will enable them to receive personalized hormonal treatment through frequent, convenient, and accurate measurements of hormone levels in peripheral blood.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

Chronic thoracic pain after cardiac surgery: role of inflammation and biomechanical sternal stability.

Introduction: The pathogenesis of chronic chest pain after cardiac surgery has not been determinate. If left untreated, postoperative sternal pain reduces the quality of life and patient satisfaction with cardiac surgery. The purpose of the study was to examine the effect of chest inflammation on postoperative pain, risk factors for chronic pain after cardiac surgery and to explore how chest reconstruction was associated with the intensity of pain. Methods: The authors performed a study of acute and chronic thoracic pain after cardiac surgery in patients with and without sternal infection and compared different techniques for chest reconstruction. 42 high-risk patients for the development of mediastinitis were included. Patients with mediastinitis received chest reconstruction (group 1). Their demographics and risk factors were matched with no-infection patients with chest reconstruction (group 2) and subjects who underwent conventional sternal closure (group 3). Chronic pain was assessed by the numeric rating scale after surgery. Results: The assessment of the incidence and intensity of chest pain at 3 months post-surgery demonstrated that 14 out of 42 patients across all groups still experienced chronic pain. Specifically, in group 1 with sternal infection five patients had mild pain, while one patient experienced mild pain in group 2, and eight patients in group 3. Also, follow-up results indicated that the highest pain score was in group 3. While baseline levels of cytokines were increased among patients with sternal infection, at discharge only the level of interleukin 6 remained high compared to no infection groups. Compared to conventional closure, after chest reconstruction, we found better healing scores at 3-month follow-up and a higher percentage of patients with the complete sternal union. Conclusions: Overall, 14 out of 42 patients have chronic pain after cardiac surgery. The intensity of the pain in mediastinitis patients significantly decreased at 3 months follow-up after chest reconstruction. Thus, post-surgery mediastinitis is not a determining factor for development the chronic chest pain. There is no correlation between cytokines levels and pain score except interleukin 6 which remains elevated for a long time after treatment. Correlation between sternal healing score and chronic chest pain was demonstrated.

Acid ceramidase gene therapy ameliorates pulmonary arterial hypertension with right heart dysfunction.

BACKGROUND: Up-regulation of ceramides in pulmonary hypertension (PH), contributing to perturbations in sphingolipid homeostasis and the transition of cells to a senescence state. We assessed the safety, feasibility, and efficiency of acid ceramidase gene transfer in a rodent PH model. METHODS: A model of PH was established by the combination of left pneumonectomy and injection of Sugen toxin. Magnetic resonance imaging and right heart catheterization confirmed development of PH. Animals were subjected to intratracheal administration of synthetic adeno-associated viral vector (Anc80L65) carrying the acid ceramidase (Anc80L65.AC), an empty capsid vector, or saline. Therapeutic efficacy was evaluated 8 weeks after gene delivery. RESULTS: Hemodynamic assessment 4 weeks after PH model the development demonstrated an increase in the mean pulmonary artery pressure to 30.4 ± 2.13 mmHg versus 10.4 ± 1.65 mmHg in sham (p < 0.001), which was consistent with the definition of PH. We documented a significant increase in pulmonary vascular resistance in the saline-treated (6.79 ± 0.85 mm Hg) and empty capsid (6.94 ± 0.47 mm Hg) groups, but not in animals receiving Anc80L65.AC (4.44 ± 0.71 mm Hg, p < 0.001). Morphometric analysis demonstrated an increase in medial wall thickness in control groups in comparison to those treated with acid ceramidase. After acid ceramidase gene delivery, a significant decrease of pro-inflammatory factors, interleukins, and senescence markers was observed. CONCLUSION: Gene delivery of acid ceramidase provided tropism to pulmonary tissue and ameliorated vascular remodeling with right ventricular dysfunction in pulmonary hypertension.

Cardiac Targeted Adeno-Associated Virus Injection in Rats.

Gene therapy is a promising approach in the treatment of cardiovascular diseases. The vectors available for cardiovascular gene therapy have significantly improved over time. Cardiac tropism is a primary characteristic of an ideal vector along with a long-term expression profile and a minimal risk of cellular immune response. Preclinical and clinical studies have demonstrated that adeno-associated viral (AAV) vectors are one of the most attractive vehicles for gene transfer. AAV has gained great popularity in the last years because of its biological properties and advantages over other viral vector systems. In this chapter we will describe methods for intracardiac delivery of AAV vector in rats.

Surgical Methods for Cardiac Gene Delivery in Large Animals.

This chapter describes main strategies of surgical gene delivery in large animals. Existing methods of cardiac gene transfer can be classified by the site of injection, interventional approach, and type of cardiac circulation at the time of transfer. Randomized clinical trials have suggested that the therapeutic benefits of gene therapy are not as substantial as expected from animal studies. This discordance in results is largely due to gene delivery methods that may be effective in small animals but are not scalable to larger species and, therefore, cannot transduce a sufficient fraction of myocytes to establish long-term clinical efficacy. Ideally, an optimized gene transfer should incorporate the following: a closed-loop recirculation for extended transgene residence time; vector washout form the vascular system after transfer to prevent collateral expression; use of methods to increase myocardial transcapillary gradient for viral particles for a better transduction, probably retrograde route of gene delivery through the coronary venous system; and myocardial ischemic preconditioning.

Efficient cardiac gene transfer and early-onset expression of a synthetic adeno-associated viral vector, Anc80L65, after intramyocardial administration.

OBJECTIVE: Gene therapy is a promising approach in the treatment of cardiovascular diseases. Preclinical and clinical studies have demonstrated that adeno-associated viral vectors are the most attractive vehicles for gene transfer. However, preexisting immunity, delayed gene expression, and postinfection immune response limit the success of this technology. The aim of this study was to investigate the efficacy of the first synthetic adeno-associated viral lineage clone, Anc80L65, for cardiac gene therapy. METHODS: By combining 2 different reporter approaches by fluorescence with green fluorescent protein and bioluminescence (Firefly luciferase), we compared transduction efficiency of Anc80L65 and adeno-associated virus, serotype 9 in neonatal rat cardiomyocytes ex vivo and rat hearts in vivo after intramyocardial and intracoronary administration. RESULTS: In cardiomyocytes, Anc80L65 provided a green fluorescent protein expression of 28.9% (36.4 ± 3.34 cells/field) at 24 hours and approximately 100% on day 7. In contrast, adeno-associated virus, serotype 9 green fluorescent protein provided minimal green fluorescent protein expression of 5.64% at 24 hours and 11.8% on day 7. After intramyocardial injection, vector expression peaked on day 7 with Anc80L65; however, with adeno-associated virus, serotype 9 the peak expression was during week 6. Administration of Anc80L65 demonstrated significantly more efficient expression of reporter gene than after adeno-associated virus, serotype 9 at 6 weeks (6.81 ± 0.64 log10 gc/100 ng DNA vs 6.49 ± 0.28 log10 gc/100 ng DNA, P < .05). These results were consistent with the amount of genome copy per cell observed in the heart. CONCLUSIONS: Anc80L65 vector allows fast and robust gene transduction compared with adeno-associated virus, serotype 9 vector in cardiac gene therapy. Anc80L65 did not adversely affect cardiac function and caused no inflammatory response or toxicity.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

BACKGROUND: Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI. METHODS: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI. RESULTS: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils. CONCLUSIONS: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.

Effects of genetic transfection on calcium cycling pathways mediated by double-stranded adeno-associated virus in postinfarction remodeling.

OBJECTIVE: Restoring calcium sensor protein (S100A1) activity in failing hearts poses a promising therapeutic strategy. We hypothesize that cardiac overexpression of the S100A1 gene mediated by a double-stranded adeno-associated virus (scAAV) results in better functional and molecular improvements compared with the single-stranded virus (ssAAV). METHODS: Heart failure was induced by coronary artery ligation. Then, intramyocardial injections of saline, AAV9 empty capsid, scAAV9.S100A1, and ssAAV9.S100A1 were performed. Ten weeks postinfarction, all rats received cardiac evaluation; serum and tissue were collected for genetic analysis, cytokine profiling, and assessments of mitochondrial function and structure. RESULTS: Overexpression of AAV9.S100A1 improved systolic and diastolic function. Compared with control, ejection fraction was greater in treated groups (54.8% vs 32.3%, P < .05). Similarly, end-diastolic volume index was significantly less in the treated group than in control (1.14 vs 1.59 mL/cm2), whereas fractional shortening was greater in treated groups than control (26% vs 38%, P < .05). Interestingly, cardiac mechanics were significantly better when treated with double-stranded virus compared with single-stranded. Quantitative polymerase chain reaction demonstrated robust transfection of myocardium with the S100A1 gene, with more infection in the self-complimentary group compared with the single-stranded group (5.68 ± 0.44 vs 4.09 ± 0.25 log10 genome copies per 100 ng of DNA; P < .0001). Concentrations of the inflammatory cytokines were elevated in the ssAAV9/S100A1 group compared with the scAAV9/S100A1. Assessment of mitochondrial respiration and morphology demonstrated that injection of self-complementary vector saved both mitochondrial structure and function. CONCLUSIONS: Gene therapy of S100A1 can prevent pathologic postmyocardial infarction remodeling and decrease inflammatory response in ischemic heart failure.

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.

RFX transcription factors are essential for hearing in mice.

Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.

Interleukin-1 deficiency prolongs ovarian lifespan in mice.

Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1ß-KO mice. IL-1α-KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α-KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α-KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1ß-KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α-KO mice. The protein and mRNA of both IL-1α and IL-1ß mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2-associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1ß, IL-6, and TNF-α in ovaries of IL-1α-KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways.

Acid ceramidase maintains the chondrogenic phenotype of expanded primary chondrocytes and improves the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ~2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Pentosan polysulfate: a novel therapy for the mucopolysaccharidoses.

BACKGROUND: Pentosan polysulfate (PPS) is an FDA-approved, oral medication with anti-inflammatory and pro-chondrogenic properties. We have previously shown that animal models of the mucopolysaccharidoses (MPS) exhibit significant inflammatory disease, contributing to cartilage degeneration. Enzyme replacement therapy (ERT) only partly reduced inflammation, and anti-TNF-alpha antibody therapy significantly enhanced clinical and pathological outcomes. Here we describe the use of PPS for the treatment of MPS type VI rats. METHODOLOGY/PRINCIPAL FINDINGS: Treatment began during prenatal development and at 1 and 6 months of age. All animals were treated until they were 9 months old. Significant reductions in the serum and tissue levels of several inflammatory markers (e.g., TNF-alpha, MIP-1alpha and RANTES/CCL5) were observed, as was reduced expression of inflammatory markers in cultured articular chondrocytes. ADAMTS-5/aggrecanase-2 levels also were reduced in chondrocytes, consistent with an elevation of serum tissue inhibitor of metalloproteinase 1. Marked improvements in motility and grooming behavior occurred, along with a reduction in eye and nasal secretions and a lessening of the tracheal deformities. MicroCT and radiographic analyses further revealed that the treated MPS skulls were longer and thinner, and that the teeth malocclusions, misalignments and mineral densities were improved. MicroCT analysis of the femurs and vertebrae revealed improvements in trabecular bone mineral densities, number and spacing in a subset of treated MPS animals. Biomechanical assessments of PPS-treated spines showed partially restored torsional behaviors, suggesting increased spinal stability. No improvements were observed in cortical bone or femur length. The positive changes in the PPS-treated MPS VI rats occurred despite glycosaminoglycan accumulation in their tissues. CONCLUSIONS: Based on these findings we conclude that PPS could be a simple and effective therapy for MPS that might provide significant clinical benefits alone and in combination with other therapies.

Construction of conditional acid ceramidase knockout mice and in vivo effects on oocyte development and fertility.

The number of resting follicles in the ovary and their successful maturation during development define the fertile female lifespan. Oocytes, enclosed within follicles, are subject to natural selection, and the majority will undergo apoptosis during prenatal life through adulthood. Our previous studies revealed high levels of the lipid hydrolase, acid ceramidase (AC), in human and mouse oocytes, follicular fluid and cumulus cells. In addition, supplementation of in vitro fertilization media with recombinant AC enhanced the survival of oocytes and preimplantation embryos. Herein we constructed and used a conditional knockout mouse model of AC deficiency (cACKO) to further investigate the role of this enzyme in oocyte survival in vivo. Immunohistochemical staining, activity assays, and western blot analysis revealed that AC expression was high in the ovaries of normal mice, particularly in the theca cells. After induction of the AC gene knockout with tamoxifen (TM), AC levels decreased in ovaries, and ceramide was correspondingly elevated. A novel immunostaining method was developed to visualize follicles at various stages, and together with light microscopic examination, the transition of the follicle from the secondary to antral stage was found to be defective in the absence of AC. Western blot analysis showed elevated BAX and PARP expression in TM-treated cACKO mouse ovaries compared to control animals. In parallel, the levels of BCL-2 and anti-Mullerian hormone, a marker of ovarian reserve, were decreased. In addition to the above, there was a significant decrease in fertility observed in the TM-treated cACKO mice. Together, these data suggest that AC plays an important role in the preservation of fertility by maintaining low ceramide levels and preventing apoptosis of theca cells, thereby promoting survival of the follicle during the transition from the secondary to antral stage.

Anti-TNF-alpha therapy enhances the effects of enzyme replacement therapy in rats with mucopolysaccharidosis type VI.

BACKGROUND: Although enzyme replacement therapy (ERT) is available for several lysosomal storage disorders, the benefit of this treatment to the skeletal system is very limited. Our previous work has shown the importance of the Toll-like receptor 4/TNF-alpha inflammatory pathway in the skeletal pathology of the mucopolysaccharidoses (MPS), and we therefore undertook a study to examine the additive benefit of combining anti-TNF-alpha therapy with ERT in a rat model of MPS type VI. METHODOLOGY/PRINCIPAL FINDINGS: MPS VI rats were treated for 8 months with Naglazyme® (recombinant human N-acetyl-galactosamine-4-sulfatase), or by a combined protocol using Naglazyme® and the rat-specific anti-TNF-alpha drug, CNTO1081. Both protocols led to markedly reduced serum levels of TNF-alpha and RANKL, although only the combined treatment reduced TNF-alpha in the articular cartilage. Analysis of cultured articular chondrocytes showed that the combination therapy also restored collagen IIA1 expression, and reduced expression of the apoptotic marker, PARP. Motor activity and mobility were improved by ERT, and these were significantly enhanced by combination treatment. Tracheal deformities in the MPS VI animals were only improved by combination therapy, and there was a modest improvement in bone length. Ceramide levels in the trachea also were markedly reduced. MicroCT analysis did not demonstrate any significant positive effects on bone microarchitecture from either treatment, nor was there histological improvement in the bone growth plates. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that combining ERT with anti-TNF-alpha therapy improved the treatment outcome and led to significant clinical benefit. They also further validate the usefulness of TNF-alpha, RANKL and other inflammatory molecules as biomarkers for the MPS disorders. Further evaluation of this combination approach in other MPS animal models and patients is warranted.