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1.
J Pathol ; 261(4): 455-464, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37792603

RESUMO

Karyomegalic interstitial nephropathy (KIN) has been reported as an incidental finding in patients with childhood cancer treated with ifosfamide. It is defined by the presence of tubular epithelial cells (TECs) with enlarged, irregular, and hyperchromatic nuclei. Cellular senescence has been proposed to be involved in kidney fibrosis in hereditary KIN patients. We report that KIN could be diagnosed 7-32 months after childhood cancer diagnosis in 6/6 consecutive patients biopsied for progressive chronic kidney disease (CKD) of unknown cause between 2018 and 2021. The morphometry of nuclear size distribution and markers for DNA damage (γH2AX), cell-cycle arrest (p21+, Ki67-), and nuclear lamina decay (loss of lamin B1), identified karyomegaly and senescence features in TECs. Polyploidy was assessed by chromosome fluorescence in situ hybridization (FISH). In all six patients the number of p21-positive TECs far exceeded the typically small numbers of truly karyomegalic cells, and p21-positive TECs contained less lysozyme, testifying to defective resorption, which explains the consistently observed low-molecular-weight (LMW) proteinuria. In addition, polyploidy of TEC was observed to correlate with loss of lysozyme staining. Importantly, in the five patients with the largest nuclei, the percentage of p21-positive TECs tightly correlated with estimated glomerular filtration rate loss between biopsy and last follow-up (R2 = 0.93, p < 0.01). We conclude that cellular senescence is associated with tubular dysfunction and predicts CKD progression in childhood cancer patients with KIN and appears to be a prevalent cause of otherwise unexplained CKD and LMW proteinuria in children treated with DNA-damaging and cell stress-inducing therapy including ifosfamide. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Neoplasias , Nefrite Intersticial , Insuficiência Renal Crônica , Humanos , Criança , Nefrite Intersticial/genética , Muramidase/genética , Ifosfamida , Hibridização in Situ Fluorescente , Neoplasias/patologia , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/complicações , Proteinúria/patologia , Rim/patologia , Biópsia , Senescência Celular , Poliploidia
2.
Kidney Int ; 102(6): 1305-1319, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35921911

RESUMO

Chronic allograft dysfunction with progressive fibrosis of unknown cause remains a major issue after kidney transplantation, characterized by ischemia-reperfusion injury (IRI). One hypothesis to account for this is that spontaneous progressive tubulointerstitial fibrosis following IRI is driven by cellular senescence evolving from a prolonged, unresolved DNA damage response (DDR). Since cellular communication network factor 2 ((CCN2), formerly called connective tissue growth factor), an established mediator of kidney fibrosis, is also involved in senescence-associated pathways, we investigated the relation between CCN2 and cellular senescence following kidney transplantation. Tubular CCN2 overexpression was found to be associated with DDR, loss of kidney function and tubulointerstitial fibrosis in both the early and the late phase in human kidney allograft biopsies. Consistently, CCN2 deficient mice developed reduced senescence and tubulointerstitial fibrosis in the late phase; six weeks after experimental IRI. Moreover, tubular DDR markers and plasma urea were less elevated in CCN2 knockout than in wild-type mice. Finally, CCN2 administration or overexpression in epithelial cells induced upregulation of tubular senescence-associated genes including p21, while silencing of CCN2 alleviated DDR induced by anoxia-reoxygenation injury in cultured proximal tubule epithelial cells. Thus, our observations indicate that inhibition of CCN2 can mitigate IRI-induced acute kidney injury, DNA damage, and the subsequent DDR-senescence-fibrosis sequence. Hence, targeting CCN2 might help to protect the kidney from transplantation-associated post-IRI chronic kidney dysfunction.


Assuntos
Injúria Renal Aguda , Fator de Crescimento do Tecido Conjuntivo , Dano ao DNA , Traumatismo por Reperfusão , Animais , Humanos , Camundongos , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose , Rim/patologia , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologia
3.
Am J Physiol Renal Physiol ; 311(5): F926-F934, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27558559

RESUMO

Age is associated with an increased prevalence of chronic kidney disease (CKD), which, through progressive tissue damage and fibrosis, ultimately leads to loss of kidney function. Although much effort is put into studying CKD development experimentally, age has rarely been taken into account. Therefore, we investigated the effect of age on the development of renal tissue damage and fibrosis in a mouse model of obstructive nephropathy (i.e., unilateral ureter obstruction; UUO). We observed that after 14 days, obstructed kidneys of old mice had more tubulointerstitial atrophic damage but less fibrosis than those of young mice. This was associated with reduced connective tissue growth factor (CTGF), and higher bone morphogenetic protein 6 (BMP6) expression and pSMAD1/5/8 signaling, while transforming growth factor-ß expression and transcriptional activity were no different in obstructed kidneys of old and young mice. In vitro, CTGF bound to and inhibited BMP6 activity. In summary, our data suggest that in obstructive nephropathy atrophy increases and fibrosis decreases with age and that this relates to increased BMP signaling, most likely due to higher BMP6 and lower CTGF expression.


Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Rim/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/fisiologia , Obstrução Ureteral/metabolismo , Fatores Etários , Animais , Proteína Morfogenética Óssea 6/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Rim/patologia , Camundongos , Fosforilação , Insuficiência Renal Crônica/patologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/patologia
4.
Growth Factors ; 34(3-4): 149-58, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27686612

RESUMO

AIMS: Connective tissue growth factor (CTGF) plays a key role in tissue fibrogenesis and growing evidence indicates a pathogenic role in cardiovascular disease. Aim of this study is to investigate the association of connective tissue growth factor (CTGF/CCN2) with cardiovascular risk and mortality in patients with manifest vascular disease. METHODS AND RESULTS: Plasma CTGF was measured by ELISA in a prospective cohort study of 1227 patients with manifest vascular disease (mean age 59.0 ± 9.9 years). Linear regression analysis was performed to quantify the association between CTGF and cardiovascular risk factors. Results are expressed as beta (ß) regression coefficients with 95% confidence intervals (CI). The relation between CTGF and the occurrence of new cardiovascular events and mortality was assessed with Cox proportional hazard analysis. Adjustments were made for potential confounding factors. Plasma CTGF was positively related to total cholesterol (ß 0.040;95%CI 0.013-0.067) and LDL cholesterol (ß 0.031;95%CI 0.000-0.062) and inversely to glomerular filtration rate (ß -0.004;95%CI -0.005 to -0.002). CTGF was significantly lower in patients with cerebrovascular disease. During a median follow-up of 6.5 years (IQR 5.3-7.4) 131 subjects died, 92 experienced an ischemic cardiac complication and 45 an ischemic stroke. CTGF was associated with an increased risk of new vascular events (HR 1.21;95%CI 1.04-1.42), ischemic cardiac events (HR 1.41;95%CI 1.18-1.67) and all-cause mortality (HR 1.18;95%CI 1.00-1.38) for every 1 nmol/L increase in CTGF. No relation was observed between CTGF and the occurrence of ischemic stroke. CONCLUSIONS: In patients with manifest vascular disease, elevated plasma CTGF confers an increased risk of new cardiovascular events and all-cause mortality.


Assuntos
Aterosclerose/sangue , Isquemia Encefálica/sangue , Fator de Crescimento do Tecido Conjuntivo/sangue , Acidente Vascular Cerebral/sangue , Idoso , Aterosclerose/epidemiologia , Aterosclerose/mortalidade , Isquemia Encefálica/epidemiologia , Isquemia Encefálica/mortalidade , Estudos de Casos e Controles , Colesterol/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/mortalidade
5.
Bioelectromagnetics ; 37(7): 471-80, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27434783

RESUMO

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Imageamento por Ressonância Magnética/instrumentação , Aceleradores de Partículas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Tolerância a Radiação , Radiometria , Raios X
6.
Nephrol Dial Transplant ; 30(8): 1291-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25916872

RESUMO

BACKGROUND: Outcome in patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (AGN) is difficult to predict. Scoring of renal biopsies has significant but limited predictive value. We investigated whether analysis of plasma and urine levels, and immunostaining of biopsies for the pro-fibrotic peptide connective tissue growth factor (CTGF), might improve prediction of renal outcome. METHODS: ANCA-positive patients were included. Renal biopsies were classified according to the AGN classification. Biopsies were stained for CTGF protein. CTGF was measured by ELISA at the time of renal biopsy in plasma and urine, and during follow-up in plasma. RESULTS: Eighty-two patients were included. CTGF staining was positive in crescentic lesions. Plasma CTGF at the time of renal biopsy was 2.4 ± 1.7 pmol/mL when compared with 0.5 ± 0.0 pmol/mL in healthy controls (P < 0.01). Plasma CTGF was associated with cellular crescents, but not when corrected for renal function. Plasma CTGF at baseline was associated with fibrous crescents in the follow-up biopsy, also after correction for renal function. Plasma CTGF at baseline predicted renal survival more accurately than the AGN classification. CONCLUSION: In AGN patients, CTGF was overexpressed in crescentic glomeruli. Baseline plasma CTGF predicted the percentage of fibrous crescents in later biopsies, and renal survival, suggesting that CTGF is involved in the cicatrization, as opposed to resolution of cellular crescents in AGN.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Cicatriz/patologia , Fator de Crescimento do Tecido Conjuntivo/sangue , Fator de Crescimento do Tecido Conjuntivo/urina , Glomerulonefrite/diagnóstico , Glomérulos Renais/patologia , Biomarcadores/sangue , Biomarcadores/urina , Biópsia , Estudos de Casos e Controles , Cicatriz/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Seguimentos , Glomerulonefrite/sangue , Glomerulonefrite/mortalidade , Glomerulonefrite/urina , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida
7.
Virchows Arch ; 484(5): 837-845, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38602559

RESUMO

The classical BCR::ABL1-negative myeloproliferative neoplasms (MPN) form a group of bone marrow (BM) diseases with the potential to progress to acute myeloid leukemia or develop marrow fibrosis and subsequent BM failure. The mechanism by which BM fibrosis develops and the factors that drive stromal activation and fibrosis are not well understood. Cellular Communication Network 2 (CCN2), also known as CTGF (Connective Tissue Growth Factor), is a profibrotic matricellular protein functioning as an important driver and biomarker of fibrosis in a wide range of diseases outside the marrow. CCN2 can promote fibrosis directly or by acting as a factor downstream of TGF-ß, the latter already known to contribute to myelofibrosis in MPN.To study the possible involvement of CCN2 in BM fibrosis in MPN, we assessed CCN2 protein expression by immunohistochemistry in 75 BM biopsies (55 × MPN and 20 × normal controls). We found variable expression of CCN2 in megakaryocytes with significant overexpression in a subgroup of 7 (13%) MPN cases; 4 of them (3 × essential thrombocytemia and 1 × prefibrotic primary myelofibrosis) showed no fibrosis (MF-0), 2 (1 × post-polycythemic myelofibrosis and 1 × primary myelofibrosis) showed moderate fibrosis (MF-2), and 1 (primary myelofibrosis) severe fibrosis (MF-3). Remarkably, CCN2 expression did not correlate with fibrosis or other disease parameters such as platelet count or thrombovascular events, neither in this subgroup nor in the whole study group. This suggests that in BM of MPN patients other, CCN2-independent pathways (such as noncanonical TGF-ß signaling) may be more important for the development of fibrosis.


Assuntos
Fator de Crescimento do Tecido Conjuntivo , Transtornos Mieloproliferativos , Mielofibrose Primária , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Mielofibrose Primária/patologia , Mielofibrose Primária/metabolismo , Pessoa de Meia-Idade , Masculino , Feminino , Idoso , Transtornos Mieloproliferativos/patologia , Transtornos Mieloproliferativos/metabolismo , Adulto , Medula Óssea/patologia , Medula Óssea/metabolismo , Idoso de 80 Anos ou mais , Imuno-Histoquímica , Fibrose/patologia
8.
J Biol Chem ; 287(27): 22854-64, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22582387

RESUMO

Granule exocytosis by cytotoxic lymphocytes is the key mechanism to eliminate virus-infected cells and tumor cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes. All human granzymes display distinct substrate specificities and induce cell death by cleaving critical intracellular death substrates. In the present study, we show that all human granzymes directly cleaved the DNA/RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K), designating hnRNP K as the first known pan-granzyme substrate. Cleavage of hnRNP K was more efficient in the presence of RNA and occurred in two apparent proteolysis-sensitive amino acid regions, thereby dissecting the functional DNA/RNA-binding hnRNP K domains. HnRNP K was cleaved under physiological conditions when purified granzymes were delivered into living tumor cells and during lymphokine-activated killer cell-mediated attack. HnRNP K is essential for tumor cell viability, since knockdown of hnRNP K resulted in spontaneous tumor cell apoptosis with caspase activation and reactive oxygen species production. This apoptosis was more pronounced at low tumor cell density where hnRNP K knockdown also triggered a caspase-independent apoptotic pathway. This suggests that hnRNP K promotes tumor cell survival in the absence of cell-cell contact. Silencing of hnRNP K protein expression rendered tumor cells more susceptible to cellular cytotoxicity. We conclude that hnRNP K is indispensable for tumor cell viability and our data suggest that targeting of hnRNP K by granzymes contributes to or reinforces the cell death mechanisms by which cytotoxic lymphocytes eliminate tumor cells.


Assuntos
Granzimas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/imunologia , Sobrevivência Celular/imunologia , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Células Jurkat , Células K562 , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Ribonucleases/farmacologia , Especificidade por Substrato , Linfócitos T Citotóxicos/enzimologia
10.
Biochem J ; 437(3): 431-42, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21564021

RESUMO

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.


Assuntos
Variação Genética , Granzimas/metabolismo , Sequência de Aminoácidos , Animais , Morte Celular , Linhagem Celular , Regulação da Expressão Gênica , Granzimas/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Conformação Proteica , Especificidade da Espécie , Especificidade por Substrato , Tubulina (Proteína)/metabolismo
11.
Front Oncol ; 11: 681967, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34178673

RESUMO

Angiogenesis is crucial for normal development and homeostasis, but also plays a role in many diseases including cardiovascular diseases, autoimmune diseases, and cancer. Granzymes are serine proteases stored in the granules of cytotoxic cells, and have predominantly been studied for their pro-apoptotic role upon delivery in target cells. A growing body of evidence is emerging that granzymes also display extracellular functions, which largely remain unknown. In the present study, we show that extracellular granzyme K (GrK) inhibits angiogenesis and triggers endothelial cells to release soluble VEGFR1 (sVEGFR1), a decoy receptor that inhibits angiogenesis by sequestering VEGF-A. GrK does not cleave off membrane-bound VEGFR1 from the cell surface, does not release potential sVEGFR1 storage pools from endothelial cells, and does not trigger sVEGFR1 release via protease activating receptor-1 (PAR-1) activation. GrK induces de novo sVEGFR1 mRNA and protein expression and subsequent release of sVEGFR1 from endothelial cells. GrK protein is detectable in human colorectal tumor tissue and its levels positively correlate with sVEGFR1 protein levels and negatively correlate with T4 intratumoral angiogenesis and tumor size. In conclusion, extracellular GrK can inhibit angiogenesis via secretion of sVEGFR1 from endothelial cells, thereby sequestering VEGF-A and impairing VEGFR signaling. Our observation that GrK positively correlates with sVEGFR1 and negatively correlates with angiogenesis in colorectal cancer, suggest that the GrK-sVEGFR1-angiogenesis axis may be a valid target for development of novel anti-angiogenic therapies in cancer.

12.
Front Immunol ; 12: 661761, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122421

RESUMO

Background: Connective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD). Materials and Methods: CTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients. Results: qPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74). Conclusions: Lung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Fator de Crescimento do Tecido Conjuntivo/genética , Expressão Gênica , Transplante de Pulmão/efeitos adversos , Pulmão/química , Fibrose Pulmonar/genética , Adulto , Feminino , Doença Enxerto-Hospedeiro/fisiopatologia , Humanos , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/etiologia , Transplante Homólogo
13.
Antioxidants (Basel) ; 10(12)2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34943123

RESUMO

AKI, due to the fact of altered oxygen supply after kidney transplantation, is characterized by renal ischemia-reperfusion injury (IRI). Recent data suggest that AKI to CKD progression may be driven by cellular senescence evolving from prolonged DNA damage response (DDR) following oxidative stress. Cellular communication factor 2 (CCN2, formerly called CTGF) is a major contributor to CKD development and was found to aggravate DNA damage and the subsequent DDR-cellular senescence-fibrosis sequence following renal IRI. We therefore investigated the impact of CCN2 inhibition on oxidative stress and DDR in vivo and in vitro. Four hours after reperfusion, full transcriptome RNA sequencing of mouse IRI kidneys revealed CCN2-dependent enrichment of several signaling pathways, reflecting a different immediate stress response to IRI. Furthermore, decreased staining for γH2AX and p-p53 indicated reduced DNA damage and DDR in tubular epithelial cells of CCN2 knockout (KO) mice. Three days after IRI, DNA damage and DDR were still reduced in CCN2 KO, and this was associated with reduced oxidative stress, marked by lower lipid peroxidation, protein nitrosylation, and kidney expression levels of Nrf2 target genes (i.e., HMOX1 and NQO1). Finally, silencing of CCN2 alleviated DDR and lipid peroxidation induced by anoxia-reoxygenation injury in cultured PTECs. Together, our observations suggest that CCN2 inhibition might mitigate AKI by reducing oxidative stress-induced DNA damage and the subsequent DDR. Thus, targeting CCN2 might help to limit post-IRI AKI.

14.
J Cell Commun Signal ; 14(1): 47-52, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32291573

RESUMO

Pulmonary fibrosis is a severely disabling disease often leading to death. CCN2 (Cellular Communication Network factor 2, also known as CTGF) is a known mediator of fibrosis and clinical trials studying anti-CCN2 efficacy in pulmonary fibrosis are currently underway. Fork head box D1 (FoxD1) transcription factor is transiently expressed in several mesenchymal cell types, including those of fetal lungs. Differentiation of FoxD1-progenitor derived pericytes into myofibroblasts involves CCN2 expression and contributes importantly to maladaptive tissue remodeling in for example kidney and lung fibrosis models. To generate a model for studying the contribution of CCN2 expression in FoxD1-progenitor derived cells to development of fibrotic tissue remodeling, we set out to establish a FoxD1Cre - CCN2flox/flox mouse colony. However, all double-transgenic mice died soon after birth due to asphyxia. Histopathological examination revealed a reduction in alveolar space and lung weight, and subtle axial (thoracic and cervical) skeletal deformities. Together with the previously reported association of a FoxD1 containing locus with human adolescent idiopathic scoliosis, our data suggest that the fatal pulmonary hypoplasia resulting from selective deletion of CCN2 from FoxD1-progenitor derived mesenchymal cells developed secondary to impaired breathing movements due to aberrant axial skeletogenesis.

15.
Int J Cancer ; 125(7): 1542-50, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19569240

RESUMO

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well-defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non-neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease-free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/química , Neoplasias de Cabeça e Pescoço/patologia , Serpinas/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/química , Neoplasias Bucais/enzimologia , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Neoplasias Orofaríngeas/química , Neoplasias Orofaríngeas/patologia , Valor Preditivo dos Testes , Prognóstico , Inibidores de Proteases/metabolismo , Serpinas/genética , Serpinas/metabolismo , Neoplasias Cutâneas/química , Neoplasias Cutâneas/patologia
16.
J Cell Commun Signal ; 13(4): 573-577, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32020419

RESUMO

Pulmonary fibrosis is a severely disabling disease often leading to death. CCN2 (Cellular Communication Network factor 2, also known as CTGF) is a known mediator of fibrosis and clinical trials studying anti-CCN2 efficacy in pulmonary fibrosis are currently underway. Fork head box D1 (FoxD1) transcription factor is transiently expressed in several mesenchymal cell types, including those of fetal lungs. Differentiation of FoxD1-progenitor derived pericytes into myofibroblasts involves CCN2 expression and contributes importantly to maladaptive tissue remodeling in e.g. kidney and lung fibrosis models. To generate a model for studying the contribution of CCN2 expression in FoxD1-progenitor derived cells to development of fibrotic tissue remodeling, we set out to establish a FoxD1Cre - CCN2flox/flox mouse colony. However, all double-transgenic mice died soon after birth due to asphyxia. Histopathological examination revealed a reduction in alveolar space and lung weight, and subtle axial (thoracic and cervical) skeletal deformities. Together with the previously reported association of a FoxD1 containing locus with human adolescent idiopathic scoliosis, our data suggest that the development of fatal pulmonary hypoplasia caused by selective deletion of CCN2 from FoxD1-progenitor derived mesenchymal cells was secondary to aberrant axial skeletogenesis.

17.
Oncoimmunology ; 7(3): e1398877, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29399402

RESUMO

Pediatric medulloblastomas are the most frequently diagnosed embryonal tumors of the central nervous system. Current therapies cause severe neurological and cognitive side effects including secondary malignancies. Cellular immunotherapy might be key to improve survival and to avoid morbidity. Efficient killing of tumor cells using immunotherapy requires to overcome cancer-associated strategies to evade cytotoxic immune responses. Here, we examined the immune response and immune evasion strategies in pediatric medulloblastomas. Cytotoxic T-cells, infiltrating medulloblastomas with variable activation status, showed no correlation with overall survival of the patients. We found limited numbers of PD1+ T-cells and complete absence of PD-L1 on medulloblastomas. Medulloblastomas downregulated immune recognition molecules MHC-I and CD1 d. Intriguingly, expression of granzyme inhibitors SERPINB1 and SERPINB4 was acquired in 23% and 50% of the tumors, respectively. Concluding, pediatric medulloblastomas exploit multiple immune evasion strategies to overcome immune surveillance. Absence of PD-L1 expression in medulloblastoma suggest limited or no added value for immunotherapy with PD1/PD-L1 blockers.

18.
J Histochem Cytochem ; 55(5): 443-52, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17164409

RESUMO

Several autoinflammatory disorders such as Muckle-Wells syndrome are characterized by mutations in the NALP3/cryopyrin gene. NALP3 and NALP1 proteins can assemble to inflammasomes that activate caspase-1, resulting in the processing of pro-inflammatory cytokines IL-1beta and IL-18. The present study was designed to determine which cells and tissues express NALP1 and NALP3. Monoclonal antibodies were developed and their use revealed distinct distribution profiles of NALP1 and NALP3. Granulocytes, monocytes (very weakly), dendritic cells, and B and T cells all express NALP1 and NALP3. Highest levels of NALP1 are found in T cells and Langerhans cells. Furthermore, NALP1 is present in glandular epithelial structures such as stomach, gut, lung, and, surprisingly, in neurons and testis. In contrast to NALP1, NALP3 shows a more restricted tissue distribution with expression mainly in non-keratinizing epithelia in the oropharynx, esophagus, and ectocervix. Moreover, NALP3 expression is found in the urothelial layer in the bladder. Likewise, a difference in subcellular distribution between NALP1 and NALP3 is observed because NALP1 is localized mainly in the nucleus, whereas NALP3 is predominantly cytoplasmic. We propose that the presence of NALP3 in epithelial cells lining the oral and genital tracts allows the rapid sensing of invading pathogens, thereby triggering an innate immune response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas de Transporte/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anticorpos Monoclonais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Humanos , Immunoblotting , Imuno-Histoquímica , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas NLR , Especificidade de Órgãos
19.
J Cell Commun Signal ; 11(2): 205-211, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28497232

RESUMO

A variety of conditional knock-out mice relying on Tamoxifen-driven ERT2/Cre -mediated recombination are available and have been used to study involvement of specific genes in kidney disease. However, recent data suggest that Tamoxifen itself might attenuate fibrosis when administered during experimental models of kidney disease. It has remained unclear whether this still applies also if kidney damage is initiated after a wash-out period has been implemented. Here we report that the commonly applied regimen of administration of 4 alternate day doses of 1mg Tamoxifen per mouse until 14 days prior to start of the actual experiment, in this case the induction of obstructive nephropathy by Unilateral Ureteral Obstruction (UUO), still attenuated fibrosis in female obstructed mouse kidneys, whereas this effect was not seen in male obstructed kidneys. Attenuation of fibrosis was accompanied by a reduction in nuclear ERα positivity despite absence of detectable levels of the active tamoxifen metabolite endoxifen throughout the UUO experiment. In conclusion, these results indicate that the Tamoxifen dosing regimen commonly applied in conditional gene targeting experiments might have prolonged confounding effects in female mice through attenuation of renal fibrosis independent of modulation of the expression of the targeted gene(s).

20.
Artigo em Inglês | MEDLINE | ID: mdl-27899032

RESUMO

BACKGROUND: We aimed to gain new insights into the pathogenesis of sporadic ALS (sALS) through a comprehensive proteomic analysis. METHODS: Protein profiles of the anterior and posterior horn in post-mortem spinal cord samples of 10 ALS patients and 10 controls were analysed using 2D-differential gel electrophoresis. The identified protein spots with statistically significant level changes and a spot ratio >2.0 were analysed by LC-MS/MS. RESULTS: In the posterior horn only 3 proteins were differentially expressed. In the anterior horn, 16 proteins with increased levels and 2 proteins with decreased levels were identified in ALS compared to controls. The identified proteins were involved in mitochondrial metabolism, calcium homeostasis, protein metabolism, glutathione homeostasis, protein transport and snRNP assembly. The two proteins with decreased levels, ATP5D and calmodulin, were validated by Western blot and immunostaining. Immunohistochemical and immunofluorescent double staining of ATP5D and synaptophysin showed that the reduction of ATP5D was most pronounced at synapses. CONCLUSIONS: We speculate that mitochondrial dysfunction in synaptic clefts could play an important role in sALS pathogenesis. A similar approach revealed decreased calmodulin expression mainly in the neuronal body and dendrites of ALS patients. These findings contribute to a deeper understanding of the disease process underlying ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , ATPases Translocadoras de Prótons/genética , Medula Espinal/patologia , Sinapses/patologia , Cálcio/metabolismo , Calmodulina/biossíntese , Calmodulina/genética , Regulação para Baixo/genética , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Homeostase/genética , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras , Proteínas/metabolismo , Proteômica , Medula Espinal/química
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