RESUMO
Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRß. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRß deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing.
Assuntos
Modelos Animais de Doenças , Isquemia , Macrófagos , Animais , Macrófagos/metabolismo , Macrófagos/imunologia , Isquemia/metabolismo , Isquemia/patologia , Isquemia/genética , Fenótipo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/lesões , Cicatrização/genética , Cicatrização/fisiologia , Camundongos Endogâmicos C57BL , Camundongos , Masculino , Membro Posterior/irrigação sanguínea , Neovascularização Fisiológica/genética , Regulação para Cima , Transcriptoma , Análise de Célula Única , Biomarcadores/metabolismo , Recuperação de Função Fisiológica , Camundongos KnockoutRESUMO
Fibrotic scar tissue formation occurs in humans and mice. The fibrotic scar impairs tissue regeneration and functional recovery. However, the origin of scar-forming fibroblasts is unclear. Here, we show that stromal fibroblasts forming the fibrotic scar derive from two populations of perivascular cells after spinal cord injury (SCI) in adult mice of both sexes. We anatomically and transcriptionally identify the two cell populations as pericytes and perivascular fibroblasts. Fibroblasts and pericytes are enriched in the white and gray matter regions of the spinal cord, respectively. Both cell populations are recruited in response to SCI and inflammation. However, their contribution to fibrotic scar tissue depends on the location of the lesion. Upon injury, pericytes and perivascular fibroblasts become activated and transcriptionally converge on the generation of stromal myofibroblasts. Our results show that pericytes and perivascular fibroblasts contribute to the fibrotic scar in a region-dependent manner.
Assuntos
Cicatriz , Fibroblastos , Fibrose , Pericitos , Traumatismos da Medula Espinal , Animais , Fibroblastos/patologia , Fibroblastos/metabolismo , Fibrose/patologia , Traumatismos da Medula Espinal/patologia , Camundongos , Pericitos/patologia , Pericitos/metabolismo , Masculino , Feminino , Cicatriz/patologia , Camundongos Endogâmicos C57BL , Células Estromais/patologiaRESUMO
Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.
Assuntos
Transportador 1 de Aminoácido Excitatório , Fibroblastos , Ereção Peniana , Pênis , Receptores Notch , Animais , Masculino , Camundongos , Circulação Sanguínea , Transportador 1 de Aminoácido Excitatório/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ereção Peniana/fisiologia , Pênis/irrigação sanguínea , Pênis/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Vasoconstrição , VasodilataçãoRESUMO
Recent advancements in human tissue analyses and animal models have revealed that fibrotic scarring is a common response to various lesions in the central nervous system (CNS). Perivascular cells within the brain or spinal cord give rise to stromal fibroblasts that form fibrotic scar tissue. In this review, we summarize the current understanding of fibrotic scar formation in different CNS lesions and evaluate published human single-cell gene expression datasets to gather information on perivascular cells. Specifically, we highlight the classification of pericytes and fibroblast subtypes and compare the marker expression of perivascular cells across different datasets.
Assuntos
Sistema Nervoso Central , Cicatriz , Animais , Humanos , Cicatriz/genética , Cicatriz/metabolismo , Cicatriz/patologia , Sistema Nervoso Central/metabolismo , Fibrose , Encéfalo/metabolismo , Pericitos/metabolismo , Fibroblastos/metabolismoRESUMO
Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.
Assuntos
Sistema Nervoso Central/patologia , Cicatriz/patologia , Pericitos/patologia , Envelhecimento/fisiologia , Animais , Astrócitos/patologia , Lesões Encefálicas Traumáticas/patologia , Isquemia Encefálica/patologia , Neoplasias Encefálicas/patologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Matriz Extracelular/metabolismo , Fibroblastos/patologia , Fibrose , Glioblastoma/patologia , Humanos , AVC Isquêmico/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/patologia , Células Estromais/patologiaRESUMO
Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237-241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.
Assuntos
Astrócitos/fisiologia , Conexina 30/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Animais , Diferenciação Celular , Conexina 30/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Fenômenos Eletrofisiológicos , Neurônios GABAérgicos/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Interneurônios/enzimologia , Proteínas Luminescentes , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Recombinação Genética , Tamoxifeno/farmacologiaRESUMO
CNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.
Assuntos
Cicatriz/patologia , Traumatismos da Medula Espinal/patologia , Animais , Axônios/fisiologia , Axônios/efeitos da radiação , Modelos Animais de Doenças , Potenciais Evocados/efeitos da radiação , Matriz Extracelular/metabolismo , Fibrose , Luz , Camundongos , Camundongos Transgênicos , Pericitos/citologia , Pericitos/metabolismo , Estimulação Luminosa , Tratos Piramidais/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Recuperação de Função Fisiológica , Regeneração , Córtex Sensório-Motor/fisiologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
PURPOSE: To profile different tyrosine kinase (TK) expression patterns in clear cell renal carcinoma (ccRCC). METHODS: We analysed mRNA expression levels of 89 receptor and non-receptor TK in corresponding cancer and normal renal tissue from 5 patients with ccRCC using the TaqMan Low-Density Array technology. In order to confirm aberrant TK expressions, a subsequent analysis of 25 ccRCC and corresponding normal renal tissues was performed, applying quantitative real-time PCR. To confirm mRNA expression levels on protein level, we studied ERBB4 and HCK using immunohistochemistry. RESULTS: A total of 12 TK were significantly upregulated in ccRCC (ABL2, FLT1, BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK, PTPRC, FYN and CSK), coherently 7 TK demonstrated a down-regulation (ERBB4, PDGFRA, NRTK3, SYK, ERBB2, FGFR3 and PTK7). These findings were validated by the utilization of RT-PCR for ABL2, FLT1 BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK and vice versa for ERBB4 and PDGFRA. Immunohistochemistry revealed ERBB4 expression to be significantly lower in ccRCC in comparison to papillary RCC, chromophobe RCC, renal oncocytoma and normal renal tissue (P < 0.001). HCK protein expression was reduced in ccRCC in contrast to papillary RCC (P < 0.001) or oncocytoma (P = 0.023), but similar to chromphobe RCC (P = 0.470), sarcomatoid RCC (P = 0.754) and normal renal tissue (P = 0.083). Neither ERBB4 nor HCK were correlated (P > 0.05) with clinical-pathological parameters. CONCLUSION: TK constitute valuable targets for pharmaceutical anti-cancer therapy. ERBB4 and HCK depict significantly lower expression levels in renal cancer tissues.
Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Proteínas Tirosina Quinases/genética , Transcriptoma , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-4RESUMO
BACKGROUND: The transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Therefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis) and lead to rapid mortality within 3-7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa. CONCLUSIONS: The transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9 known to be expressed in intestinal and hepatic stem/progenitor cell populations.
Assuntos
Enteropatias/metabolismo , Falência Hepática/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Enteropatias/etiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Falência Hepática/etiologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , PPAR alfa/metabolismo , Fosfatidato Fosfatase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-2/genéticaRESUMO
OBJECTIVE: To investigate the pattern of DNA CpG island hypermethylation in papillary renal cell carcinoma (pRCC). MATERIAL AND METHODS: DNA from pRCC (n= 32) and adjacent normal tissue (n= 15) was isolated. A quantitative methylation-specific PCR was performed to analyse the methylation pattern at APC (actin beta), CDH1 (E-cadherin), GSTP1 (glutathione S-transferase pi 1), RASSF1A (Ras association domain family member 1A) and TIMP3 (TIMP metallopeptidase inhibitor 3); a sequence of ACTB without CpG was used to normalize for DNA input and to calculate the relative amount of methylated DNA (normalized index of methylation, NIM). RESULTS: RASSF1A hypermethylation was observed in most pRCC and normal samples (100 vs 94.4%), but the median NIM was significantly higher in pRCC samples (2.11 vs 0.61; P < 0.001). RASSF1A hypermethylation allowed discrimination of pRCC and normal tissue with a sensitivity of 87.5% and a specificity of 73.3% as determined via receiver operator characteristic analysis (area under curve = 0.814). Hypermethylation at APC (3.0 vs 6.7%), CDH1 (15.6 vs 0%), GSTP1 (21.9 vs 6.7%) and TIMP3 (6.3 vs 0%) was infrequent in pRCC and normal tissue. CDH1 was significantly correlated with pathological stage (P= 0.015), and patients with methylated CDH1 methylation showed a trend towards shorter recurrence-free survival (log-rank P= 0.057). The number of methylated gene sites was correlated with pathological stage (P= 0.007) and lymph node metastasis (P= 0.008). CONCLUSIONS: DNA hypermethylation at RASSF1A is common in pRCC tissue irrespective of the histological subtype, but also frequently seen at lower levels in normal adjacent tissue. Aberrant hypermethylation could be a prognostic marker for pRCC.