Differential Expression of ATM, NF-KB, PINK1 and Foxo3a in Radiation-Induced Basal Cell Carcinoma.

Research in normal tissue radiobiology is in continuous progress to assess cellular response following ionizing radiation exposure especially linked to carcinogenesis risk. This was observed among patients with a history of radiotherapy of the scalp for ringworm who developed basal cell carcinoma (BCC). However, the involved mechanisms remain largely undefined. We performed a gene expression analysis of tumor biopsies and blood of radiation-induced BCC and sporadic patients using reverse transcription-quantitative PCR. Differences across groups were assessed by statistical analysis. Bioinformatic analyses were conducted using miRNet. We showed a significant overexpression of the FOXO3a, ATM, P65, TNF-α and PINK1 genes among radiation-induced BCCs compared to BCCs in sporadic patients. ATM expression level was correlated with FOXO3a. Based on receiver-operating characteristic curves, the differentially expressed genes could significantly discriminate between the two groups. Nevertheless, TNF-α and PINK1 blood expression showed no statistical differences between BCC groups. Bioinformatic analysis revealed that the candidate genes may represent putative targets for microRNAs in the skin. Our findings may yield clues as to the molecular mechanism involved in radiation-induced BCC, suggesting that deregulation of ATM-NF-kB signaling and PINK1 gene expression may contribute to BCC radiation carcinogenesis and that the analyzed genes could represent candidate radiation biomarkers associated with radiation-induced BCC.

Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.

Immunity in the Progeroid Model of Cockayne Syndrome: Biomarkers of Pathological Aging.

Cockayne syndrome (CS) is a rare autosomal recessive disorder that affects the DNA repair process. It is a progeroid syndrome predisposing patients to accelerated aging and to increased susceptibility to respiratory infections. Here, we studied the immune status of CS patients to determine potential biomarkers associated with pathological aging. CS patients, as well as elderly and young, healthy donors, were enrolled in this study. Complete blood counts for patients and donors were assessed, immune cell subsets were analyzed using flow cytometry, and candidate cytokines were analyzed via multi-analyte ELISArray kits. In CS patients, we noticed a high percentage of lymphocytes, an increased rate of intermediate and non-classical monocytes, and a high level of pro-inflammatory cytokine IL-8. In addition, we identified an increased rate of particular subtypes of T Lymphocyte CD8+ CD28- CD27-, which are senescent T cells. Thus, an inflammatory state was found in CS patients that is similar to that observed in the elderly donors and is associated with an immunosenescence status in both groups. This could explain the CS patients' increased susceptibility to infections, which is partly due to an aging-associated inflammation process.

Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.

Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Growing rods for early-onset scoliosis in Ehlers-Danlos disease.

PURPOSE: To study the results and complications of Traditional Growing Rods (TGR) for the treatment of Early-Onset Scoliosis (EOS) in patients with Ehlers-Danlos syndrome (EDS). METHODS: This is a retrospective study of patients with EDS treated for EOS between 2012 and 2018 by TGR. For each patient, we evaluated the evolution of the Cobb angle of the main coronal curve before the first surgery, postoperatively and at the last follow-up. We also noted every complication. RESULTS: Four patients have been evaluated. Age at the index surgery ranged from 3- to 6-year-old. All the patients had a Kyphoscoliosis. The preoperative Cobb angle of the main coronal curve ranged from 70° to 104°. Prior to surgery, a progressive correction of the spinal deformity by Halo-gravity traction was performed for all patients. One patient had several complications with a poor result after final fusion. Three patients did not sustain any complication and presented good results. Follow-up ranged from 3 to 8 years. For two of our patients, further lengthening procedures are still needed. CONCLUSIONS: TGR is a good surgical option for the treatment of EOS in EDS. A progressive preoperative correction is recommended. Anchor-related complications must be minimized in these patients with an important kyphosis. Level of evidence Level IV.

Case report: Exome sequencing revealed disease-causing variants in a patient with spondylospinal thoracic dysostosis.

Background: Spondylocostal dysostosis is a rare genetic disorder caused by mutations in DLL3, MESP2, LFNG, HES7, TBX6, and RIPPLY2. A particular form of this disorder characterized by the association of spondylocostal dysostosis with multiple pterygia has been reported and called spondylospinal thoracic dysostosis. Both disorders affect the spine and ribs, leading to abnormal development of the spine. Spondylospinal thoracic dysostosis is a rare syndrome characterized by the association of multiple vertebral segmentation defects, thoracic cage deformity, and multiple pterygia. This syndrome can be considered a different form of the described spondylocostal dysostosis. However, no genetic testing has been conducted for this rare disorder so far. Methods: We report here the case of an 18-month-old female patient presenting the clinical and radiological features of spondylospinal thoracic dysostosis. To determine the underlying genetic etiology, whole exome sequencing (WES) and Sanger sequencing were performed. Results: Using WES, we identified a variant in the TPM2 gene c. 628C>T, already reported in the non-lethal form of multiple pterygium syndrome. In addition, following the analysis of WES data, using bioinformatic tools, for oligogenic diseases, we identified candidate modifier genes, CAP2 and ADCY6, that could impact the clinical manifestations. Conclusion: We showed a potential association between TPM2 and the uncommon spondylocostal dysostosis phenotype that would require further validation on larger cohort.

A new model of experimental fibrosis in hindlimb skeletal muscle of adult mdx mouse mimicking muscular dystrophy.

INTRODUCTION: Duchenne Muscular Dystrophy (DMD) is characterized by the lack of dystrophin that leads to severe myofiber degeneration. We have shown that endomysial fibrosis is correlated with age at ambulation loss in DMD patients. However, the dystrophin-deficient mdx mouse does not have fibrotic lesions in adult limb muscles. Here, we describe a model of chronic mechanical muscle injury that triggers chronic lesions in mdx hindlimb muscle. METHODS: Micromechanical injuries were performed daily in tibialis anterior muscles for 2 weeks. RESULTS: Endomysial fibrosis appeared beginning 1 week post-injury, remained stable for 3 months and was associated with loss of specific maximal force. Fibrosis was associated with an increased expression of factors involved in fibrogenesis including α-smooth muscle actin, connective tissue growth factor, and lysyl oxidase, which colocalized with collagen deposits. CONCLUSIONS: This induced fibrotic dystrophic model may be useful to study mechanisms of fibrosis in dystrophinopathies and to evaluate antifibrotic treatments.

Inflammatory landscape in Xeroderma pigmentosum patients with cutaneous melanoma.

Xeroderma pigmentosum (XP) is a DNA repair disease that predisposes to early skin cancers as cutaneous melanoma. Melanoma microenvironment contains inflammatory mediators, which would be interesting biomarkers for the prognosis or for the identification of novel therapeutic targets. We used a PCR array to evaluate the transcriptional pattern of 84 inflammatory genes in melanoma tumors obtained from XP patients (XP-Mel) and in sporadic melanoma (SP-Mel) compared to healthy skin. Commonly expressed inflammatory genes were further explored via GTEx and GEPIA databases. The differentially expressed inflammatory genes in XP were compared to their expression in skin exposed to UVs, and evaluated on the basis of the overall survival outcomes of patients with melanoma. Monocyte subsets of patients with SP-Mel, XP and healthy donors were also assessed. PCR array data revealed that 34 inflammatory genes were under-expressed in XP-Mel compared to SP-Mel. Differentially expressed genes that were common in XP-Mel and SP-Mel were correlated with the transcriptomic datasets from GEPIA and GTEx and highlighted the implication of KLK1 and IL8 in the tumorigenesis. We showed also that in XP-Mel tumors, there was an overexpression of KLK6 and KLK10 genes, which seems to be associated with a bad survival rate. As for the innate immunity, we observed a decrease of intermediate monocytes in patients with SP-Mel and in XP. We highlight an alteration in the immune response in XP patients. We identified candidate biomarkers involved in the tumorigenesis, and in the survival of patients with melanoma. Intermediate monocyte's in patients at risk could be a prognostic biomarker for melanoma outcome.

Combining Gene Mutation with Expression of Candidate Genes to Improve Diagnosis of Escobar Syndrome.

Escobar syndrome is a rare, autosomal recessive disorder that affects the musculoskeletal system and the skin. Mutations in the CHRNG and TPM2 genes are associated with this pathology. In this study, we conducted a clinical and genetic investigation of five patients and also explored via in silico and gene expression analysis their phenotypic variability. In detail, we identified a patient with a novel composite heterozygous variant of the CHRNG gene and two recurrent mutations in both CHRNG and TPM2 in the rest of the patients. As for the clinical particularities, we reported a list of modifier genes in a patient suffering from myopathy. Moreover, we identified decreased expression of IGF-1, which could be related to the short stature of Escobar patients, and increased expression of POLG1 specific to patients with TPM2 mutation. Through this study, we identified the genetic spectrum of Escobar syndrome in the Tunisian population, which will allow setting up genetic counseling and prenatal diagnosis for families at risk. In addition, we highlighted relevant biomarkers that could differentiate between patients with different genetic defects.

Heterogeneous clinical features in Cockayne syndrome patients and siblings carrying the same CSA mutations.

BACKGROUND: Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in the transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and that cannot be anticipated in the diagnostic phase. In addition, little data is available for affected siblings, and this disease is largely undiagnosed in North Africa. METHODS: We report here the clinical description as well as genetic and functional characterization of eight Tunisian CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in five families, and targeted gene sequencing was done for one patient of the sixth family. We also performed Recovery RNA Synthesis (RRS) to confirm the functional impairment of DNA repair in patient-derived fibroblasts. RESULTS: Six out of eight patients carried a homozygous indel mutation (c.598_600delinsAA) in exon 7 of ERCC8, and displayed a variable clinical spectrum including between siblings sharing the same mutation. The other two patients were siblings who carried a homozygous splice-site variant in ERCC8 (c.843+1G>C). This last pair presented more severe clinical manifestations, which are rarely associated with CSA mutations, leading to gastrostomy and hepatic damage. Impaired TC-NER was confirmed by RRS in six tested patients. CONCLUSIONS: This study provides the first deep characterization of case series of CS patients carrying CSA mutations in North Africa. These mutations have been described only in this region and in the Middle-East. We also provide the largest characterization of multiple unrelated patients, as well as siblings, carrying the same mutation, providing a framework for dissecting elusive genotype-phenotype correlations in CS.

Apolipoprotein E-deficient mice develop an anti-Chlamydophila pneumoniae T helper 2 response and resist vascular infection.

BACKGROUND: Hypercholesterolemia could inhibit the immune response against various pathogens. No information is available about its impact on the immune response toward Chlamydophila pneumoniae. METHODS: Apolipoprotein E (apoE)-deficient and wild-type mice fed a normal diet were infected with a single intranasal inoculation of viable C. pneumoniae. RESULTS: Whereas interferon gamma concentrations (T helper 1 response) were similar in the lungs and spleen of apoE-deficient and wild-type mice, increased concentrations of interleukin 10, interleukin 6, and interleukin 4 (T helper 2 response) were found in the lungs of apoE-deficient mice. The spleen B lymphocyte percentage and interleukin 4 levels and serum specific antibody titers were higher in apoE-deficient mice. C. pneumoniae infection was facilitated neither in the lungs nor in the aorta of these mice. On the contrary, the number of apoE-deficient mice with detectable levels of bacterial DNA in the aorta was clearly decreased. When low-density lipoprotein receptor-deficient mice fed a normal diet were similarly infected, no difference in the interleukin 4 concentration and infection level was observed in the lungs and no protection was found in the aorta. CONCLUSIONS: Mild hypercholesterolemia in mice does not facilitate C. pneumoniae persistence in the vascular wall. ApoE deficiency, rather than mild hypercholesterolemia, probably favors the development of an unusual anti-C. pneumoniae T helper 2 response and protects against vascular infection.

Case Report: Identification of Novel Variants in ERCC4 and DDB2 Genes in Two Tunisian Patients With Atypical Xeroderma Pigmentosum Phenotype.

Xeroderma Pigmentosum (XP) is a rare genetic disorder affecting the nucleotide excision repair system (NER). It is characterized by an extreme sensitivity to sunlight that induces cutaneous disorders such as severe sunburn, freckling and cancers. In Tunisia, six complementation groups have been already identified. However, the genetic etiology remains unknown for several patients. In this study, we investigated clinical characteristics and genetic defects in two families with atypical phenotypes originating from the central region in Tunisia. Clinical investigation revealed mild cutaneous features in two patients who develop multiple skin cancers at later ages, with no neurological disorders. Targeted gene sequencing revealed that they carried novel variants. A homozygous variation in the ERCC4 gene c.1762G>T, p.V588F, detected in patient XP21. As for patient XP134, he carried two homozygous mutations in the DDB2 gene c.613T>C, p.C205R and c.618C>A, p.S206R. Structural modeling of the protein predicted the identified ERCC4 variant to mildly affect protein stability without affecting its functional domains. As for the case of DDB2 double mutant, the second variation seems to cause a mild effect on the protein structure unlike the first variation which does not seem to have an effect on it. This study contributes to further characterize the mutation spectrum of XP in Tunisian families. Targeted gene sequencing accelerated the identification of rare unexpected genetic defects for diagnostic testing and genetic counseling.

Identification and Characterization of a Novel Recurrent ERCC6 Variant in Patients with a Severe Form of Cockayne Syndrome B.

Cockayne syndrome (CS) is a rare disease caused by mutations in ERCC6/CSB or ERCC8/CSA. We report here the clinical, genetic, and functional analyses of three unrelated patients mutated in ERCC6/CSB with a severe phenotype. After clinical examination, two patients were investigated via next generation sequencing, targeting seventeen Nucleotide Excision Repair (NER) genes. All three patients harbored a novel, c.3156dup, homozygous mutation located in exon 18 of ERCC6/CSB that affects the C-terminal region of the protein. Sanger sequencing confirmed the mutation and the parental segregation in the three families, and Western blots showed a lack of the full-length protein. NER functional impairment was shown by reduced recovery of RNA synthesis with proficient unscheduled DNA synthesis after UV-C radiations in patient-derived fibroblasts. Despite sharing the same mutation, the clinical spectrum was heterogeneous among the three patients, and only two patients displayed clinical photosensitivity. This novel ERCC6 variant in Tunisian patients suggests a founder effect and has implications for setting-up prenatal diagnosis/genetic counselling in North Africa, where this disease is largely undiagnosed. This study reveals one of the rare cases of CS clinical heterogeneity despite the same mutation. Moreover, the occurrence of an identical homozygous mutation, which either results in clinical photosensitivity or does not, strongly suggests that this classic CS symptom relies on multiple factors.

Clinical and Genetic Heterogeneity in Six Tunisian Families With Horizontal Gaze Palsy With Progressive Scoliosis: A Retrospective Study of 13 Cases.

Background: Horizontal Gaze Palsy with Progressive Scoliosis (HGPPS) is a rare autosomal recessive congenital disorder characterized by the absence of conjugate horizontal eye movements, and progressive debilitating scoliosis during childhood and adolescence. HGPPS is associated with mutations of the ROBO3 gene. In this study, the objective is to identify pathogenic variants in a cohort of Tunisian patients with HGPPS and to further define ROBO3 genotype-phenotype correlations. Methods: Thirteen Tunisian patients from six unrelated consanguineous families all manifesting HGPPS were genetically investigated. We searched for the causative variants for HGPPS using classical Sanger and whole exome sequencing. Results: Four distinct homozygous mutations were identified in ROBO3 gene. Two of these were newly identified homozygous and non-synonymous mutations, causing effectively damage to the protein by in silico analysis. The other two mutations were previously reported in Tunisian patients with HGPPS. Mutations were validated by Sanger sequencing in parents and affected individuals. Conclusion: To the best of our knowledge, this is the largest ever reported cohort on families with HGPPS in whom ROBO3 mutations were identified. These molecular findings have expanded our knowledge of the ROBO3 mutational spectrum. The relevance of our current study is two-fold; first to assist proper management of the scoliosis and second to protect families at risk.

Genetic diversity and functional effect of common polymorphisms in genes involved in the first heterodimeric complex of the Nucleotide Excision Repair pathway.

Nucleotide excision repair is a multistep process that recognizes and eliminates a spectrum of DNA damages. Five proteins, namely XPC, RAD23, Centrin 2, DDB1 and DDB2 act as a heterodimeric complex at the early steps of the NER pathway and play a crucial role in the removal of DNA lesions. Several exonic mutations on genes coding for these proteins have been identified as associated with Xeroderma-pigmentosum (XP), a rare monogenic disorder. However, the role of regulatory polymorphisms in disease development and inter-ethnic diversity is still not well documented. Due to the high incidence rate of XP in Tunisia, we performed a genotyping analysis of 140 SNPs found on these 5 genes in a set of 135-subjects representing the general Tunisian-population. An inter-ethnic comparison based on the genotype frequency of these SNPs have been also conducted. For the most relevant variants, we performed a comprehensive assessment of their functional effects. Linkage disequilibrium and principal component analysis showed that the Tunisian-population is an admixed and intermediate population between Sub-Saharan Africans and Europeans. Using variable factor maps, we identified a list of 20 polymorphisms that contribute considerably to the inter-ethnic diversity of the NER complex. In-silico functional analysis showed that SNPs on XPC, DDB1 and DDB2 are associated with eQTLs mainly DDB2-rs10838681 that seems to decrease significantly the expression level of ACP2 (p = 6.1 × 10-26). Statistical analysis showed that the allelic frequency of DDB2-rs10838681 in Tunisia is significantly different from all other populations. Using rVarBase, we identified 5 variants on XPC, DDB1 and DDB2 that seem to alter the binding sites of several transcription factors considered as key players in DNA-repair pathways. Results presented in this study provide the first report on regulatory polymorphisms of the NER-complex genes in Tunisia. These results may also help to establish a baseline database for future association and functional studies.

Dual and beneficial roles of macrophages during skeletal muscle regeneration.

Macrophages are necessary for skeletal muscle regeneration after injury. Muscle recruits inflammatory monocytes/macrophages that switch toward an anti-inflammatory profile upon phagocytosis of debris. In vitro, proinflammatory macrophages stimulate myoblast proliferation, whereas anti-inflammatory macrophages stimulate their differentiation. Thus, macrophages are involved in both phases of skeletal muscle regeneration: first, inflammation and cleansing of necrosis, and then myogenic differentiation and tissue repair.

Identification of a ERCC5 c.2333T>C (L778P) Variant in Two Tunisian Siblings With Mild Xeroderma Pigmentosum Phenotype.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder due to a defect in the nucleotide excision repair (NER) DNA repair pathway, characterized by severe sunburn development of freckles, premature skin aging, and susceptibility to develop cancers at an average age of eight. XP is an example of accelerated photo-aging. It is a genetically and clinically heterogeneous disease. Eight complementation groups have been described worldwide. In Tunisia, five groups have been already identified. In this work, we investigated the genetic etiology in a family with an atypically mild XP phenotype. Two Tunisian siblings born from first-degree consanguineous parents underwent clinical examination in the dermatology department of the Charles Nicolle Hospital on the basis of acute sunburn reaction and mild neurological disorders. Blood samples were collected from two affected siblings after written informed consent. As all mutations reported in Tunisia have been excluded using Sanger sequencing, we carried out mutational analysis through a targeted panel of gene sequencing using the Agilent HaloPlex target enrichment system. Our clinical study shows, in both patients, the presence of achromic macula in sun exposed area with dermatological feature suggestive of Xeroderma pigmentosum disease. No developmental and neurological disorders were observed except mild intellectual disability. Genetic investigation shows that both patients were carriers of an homozygous T to C transition at the nucleotide position c.2333, causing the leucine to proline amino acid change at the position 778 (p.Leu778Pro) of the ERCC5 gene, and resulting in an XP-G phenotype. The same variation was previously reported at the heterozygous state in a patient cell line in Europe, for which no clinical data were available and was suggested to confer an XP/CS phenotype based on functional tests. This study contributes to further characterization of the mutation spectrum of XP in consanguineous Tunisian families and is potentially helpful for early diagnosis. It also indicates that the genotype-phenotype correlation is not always coherent for patients with mild clinical features. These data therefore suggest that targeted NGS is a highly informative diagnostic strategy, which can be used for XP molecular etiology determination.

A Tunisian family with a novel mutation in the gene CYP4F22 for lamellar ichthyosis and co-occurrence of hearing loss in a child due to mutation in the SLC26A4 gene.

BACKGROUND: Co-occurrence of two genetic diseases is challenging for accurate diagnosis and genetic counseling. The recent availability of whole exome sequencing (WES) has dramatically improved the molecular diagnosis of rare genetic diseases in particular in consanguineous populations. METHODS: We report here on a consanguineous family from Southern Tunisia including three members affected with congenital ichthyosis. The index case had a hearing loss (HL) and ichthyosis and was primarily suspected as suffering from keratitis-ichthyosis-deafness (KID) syndrome. WES was performed for the index case, and all members of the nuclear family were sequenced (Sanger method). RESULTS: The WES approach allowed the identification of two strong candidate variants in two different genes; a missense mutation c.1334T>G (p.Leu445Trp) in exon 11 of SLC26A4 gene, associated with isolated HL and a novel missense mutation c.728G>T (p.Arg243Leu) in exon 8 of CYP4F22 gene likely responsible for ichthyosis. These two mutations were predicted to be pathogenic by three pathogenicity prediction softwares (Scale-Invariant Feature Transform [SIFT], Polymorphism Phenotyping [PolyPhen], Mutation Taster) to underlie the HL and ichthyosis, respectively. CONCLUSIONS: The present study raises awareness about the importance of familial history for accurate diagnosis of syndromic genetic diseases and differential diagnosis with co-occurrence of two distinct clinical entities. In addition, in countries with limited resources, WES sequencing for a single individual provides a cost effective tool for molecular diagnosis confirmation and genetic counseling.

Antiangiogenic treatment prevents adventitial constrictive remodeling in graft arteriosclerosis.

BACKGROUND: Lumen loss in graft arteriosclerosis is the consequence of the development of a thick neointima and constrictive arterial remodeling. The latter is due to adventitial chronic inflammation and excessive perivascular collagen deposition. We reasoned that blockade of the portal of entry of inflammatory effectors may constitute a strategy to prevent constrictive arterial remodeling. METHODS AND RESULTS: We found that an anti-angiogenic therapy (ABT-510 nonapeptide), devoid of direct immunomodulatory properties, dramatically reduced adventitial angiogenesis by 66% (P<0.0001) in the rat aortic interposition model of graft arteriosclerosis. The associated decreased entry of inflammatory cells (44%; P<0.00001) resulted in drastic reduction of collagen deposition (57%; P<0.0001) thereby preventing subsequent adventitial constrictive remodeling and reduction of lumen surface area (5.26+/-0.74 vs. 8.58+/-2.48 microm2; Control vs. ABT-510-treated rats; P<0.0001). ABT-510 had no effect on the development of the neointima. CONCLUSION: This work supports the idea that targeting angiogenesis may act synergistically with conventional immunosuppressive therapy in preventing graft arteriosclerosis, a crucial feature of chronic graft rejection.

AMPK Activation Regulates LTBP4-Dependent TGF-ß1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy.

Chronic inflammation and fibrosis characterize Duchenne muscular dystrophy (DMD). We show that pro-inflammatory macrophages are associated with fibrosis in mouse and human DMD muscle. DMD-derived Ly6Cpos macrophages exhibit a profibrotic activity by sustaining fibroblast production of collagen I. This is mediated by the high production of latent-TGF-ß1 due to the higher expression of LTBP4, for which polymorphisms are associated with the progression of fibrosis in DMD patients. Skewing macrophage phenotype via AMPK activation decreases ltbp4 expression by Ly6Cpos macrophages, blunts the production of latent-TGF-ß1, and eventually reduces fibrosis and improves DMD muscle force. Moreover, fibro-adipogenic progenitors are the main providers of TGF-ß-activating enzymes in mouse and human DMD, leading to collagen production by fibroblasts. In vivo pharmacological inhibition of TGF-ß-activating enzymes improves the dystrophic phenotype. Thus, an AMPK-LTBP4 axis in inflammatory macrophages controls the production of TGF-ß1, which is further activated by and acts on fibroblastic cells, leading to fibrosis in DMD.