RESUMEN
Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.
Asunto(s)
Calcinosis , Catarata , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Glucosa , Hiperglucemia , Factor 1 Inducible por Hipoxia , Cristalino , Humanos , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/genética , Calcinosis/etiología , Calcinosis/metabolismo , Calcinosis/patología , Catarata/etiología , Catarata/metabolismo , Catarata/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Glucosa/metabolismo , Hiperglucemia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cristalino/metabolismo , Cristalino/patología , Osteocalcina/metabolismo , Osteocalcina/genética , Transducción de Señal , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.
Asunto(s)
Factor XIIIa , Placa Aterosclerótica , Transglutaminasas , Colágeno , Células Endoteliales/metabolismo , Factor XIIIa/genética , Factor XIIIa/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/metabolismo , Trombospondina 1/genética , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
Natterin is a potent pro-inflammatory fish molecule, inducing local and systemic IL-1ß/IL-1R1-dependent neutrophilia mediated by non-canonical NLRP6 and NLRC4 inflammasome activation in mice, independent of NLRP3. In this work, we investigated whether Natterin activates mitochondrial damage, resulting in self-DNA leaks into the cytosol, and whether the DNA sensor cGAS and STING pathway participate in triggering the innate immune response. Employing a peritonitis mouse model, we found that the deficiency of the tlr2/tlr4, myd88 and trif results in decreased neutrophil influx to peritoneal cavities of mice, indicative that in addition to MyD88, TRIF contributes to neutrophilia triggered by TLR4 engagement by Natterin. Next, we demonstrated that gpcr91 deficiency in mice abolished the neutrophil recruitment after Natterin injection, but mice pre-treated with 2-deoxy-d-glucose that blocks glycolysis presented similar infiltration than WT Natterin-injected mice. In addition, we observed that, compared with the WT Natterin-injected mice, DPI and cyclosporin A treated mice had a lower number of neutrophils in the peritoneal exudate. The levels of dsDNA in the supernatant of the peritoneal exudate and processed IL-33 in the supernatant of the peritoneal exudate or cytoplasmic supernatant of the peritoneal cell lysate of WT Natterin-injected mice were several folds higher than those of the control mice. The recruitment of neutrophils to peritoneal cavity 2 h post-Natterin injection was intensely impaired in ifnar KO mice and partially in il-28r KO mice, but not in ifnγr KO mice. Finally, using cgas KO, sting KO, or irf3 KO mice we found that recruitment of neutrophils to peritoneal cavities was virtually abolished in response to Natterin. These findings reveal cytosolic DNA sensors as critical regulators for Natterin-induced neutrophilia.
Asunto(s)
Factor 88 de Diferenciación Mieloide , Receptor Toll-Like 4 , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , ADN , Venenos de los Peces , Proteínas de la Membrana/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas Citotóxicas Formadoras de Poros , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
Ornamental aquaculture and the related pet industry are known to be important sources of non-native species worldwide. In the temperate zone, thermal waterbodies are attractive places for irresponsible owners to release unwanted freshwater pets including decapod crustaceans. Several non-native ornamental species have been reported in the thermal locality of Miskolctapolca (a suburb of Miskolc, Hungary). So we surveyed this site in March 2019-November 2021 to update local occurrence records and detect potentially newly released species. A well-established population of Neocaridina denticulata and the occurrence of Caridina cf. babaulti had previously been noted. However, for the first time at this site, we found the shrimps Atyopsis moluccensis, Caridina gracilirostris and C. multidentata, as well as the crayfish Procambarus virginalis, P. clarkii, Cherax quadricarinatus, C. boesemani and C. snowden, and several formally undescribed Cherax species originating from New Guinea. Furthermore, in most species, gravid females carrying eggs were also noticed. Three shrimps, A. moluccensis, C. gracilirostris and C. multidentata, were recorded for the first time in European wild. Further monitoring of this locality and better education of the general public regarding the risks associated with the release of non-native species are strongly recommended.
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Astacoidea , Decápodos , Animales , Monitoreo del Ambiente , Femenino , Agua Dulce , HungríaRESUMEN
Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.
Asunto(s)
Hemorragia Cerebral/patología , Plexo Coroideo/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/patología , Proteína C-Reactiva/líquido cefalorraquídeo , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/congénito , Hemorragia Cerebral/metabolismo , Plexo Coroideo/patología , Estudios de Cohortes , Citocinas/líquido cefalorraquídeo , Citocinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Hemo/metabolismo , Hemoglobinas/metabolismo , Humanos , Hungría , Recién Nacido , Recien Nacido Prematuro , Molécula 1 de Adhesión Intercelular/líquido cefalorraquídeo , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Síndrome de Respuesta Inflamatoria Sistémica/congénito , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Molécula 1 de Adhesión Celular Vascular/líquido cefalorraquídeo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Objective- Vascular calcification is associated with high risk of cardiovascular events and mortality. Osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) is the major cellular mechanism underlying vascular calcification. Because tissue hypoxia is a common denominator in vascular calcification, we investigated whether hypoxia per se triggers osteochondrogenic differentiation of VSMCs. Approach and Results- We studied osteochondrogenic differentiation of human aorta VSMCs cultured under normoxic (21% O2) and hypoxic (5% O2) conditions. Hypoxia increased protein expression of HIF (hypoxia-inducible factor)-1α and its target genes GLUT1 (glucose transporter 1) and VEGFA (vascular endothelial growth factor A) and induced mRNA and protein expressions of osteochondrogenic markers, that is, RUNX2 (runt-related transcription factor 2), SOX9 (Sry-related HMG box-9), OCN (osteocalcin) and ALP (alkaline phosphatase), and induced a time-dependent calcification of the extracellular matrix of VSMCs. HIF-1 inhibition by chetomin abrogated the effect of hypoxia on osteochondrogenic markers and abolished extracellular matrix calcification. Hypoxia triggered the production of reactive oxygen species, which was inhibited by chetomin. Scavenging reactive oxygen species by N-acetyl cysteine attenuated hypoxia-mediated upregulation of HIF-1α, RUNX2, and OCN protein expressions and inhibited extracellular matrix calcification, which effect was mimicked by a specific hydrogen peroxide scavenger sodium pyruvate and a mitochondrial reactive oxygen species inhibitor rotenone. Ex vivo culture of mice aorta under hypoxic conditions triggered calcification which was inhibited by chetomin and N-acetyl cysteine. In vivo hypoxia exposure (10% O2) increased RUNX2 mRNA levels in mice lung and the aorta. Conclusions- Hypoxia contributes to vascular calcification through the induction of osteochondrogenic differentiation of VSMCs in an HIF-1-dependent and mitochondria-derived reactive oxygen species-dependent manner.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factor 1 Inducible por Hipoxia/genética , Hipoxia/complicaciones , Especies Reactivas de Oxígeno/metabolismo , Calcificación Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Disulfuros/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Alcaloides Indólicos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/genética , Distribución Aleatoria , Valores de Referencia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Calcificación Vascular/fisiopatologíaRESUMEN
Osteogenic differentiation of multipotent mesenchymal stem cells (MSCs) plays a crucial role in bone remodeling. Numerous studies have described the deleterious effect of iron overload on bone density and microarchitecture. Excess iron decreases osteoblast activity, leading to impaired extracellular matrix (ECM) mineralization. Additionally, iron overload facilitates osteoclast differentiation and bone resorption. These processes contribute to iron overload-associated bone loss. In this study we investigated the effect of iron on osteogenic differentiation of human bone marrow MSCs (BMSCs), the third player in bone remodeling. We induced osteogenic differentiation of BMSCs in the presence or absence of iron (0-50µmol/L) and examined ECM mineralization, Ca content of the ECM, mRNA and protein expressions of the osteogenic transcription factor runt-related transcription factor 2 (Runx2), and its targets osteocalcin (OCN) and alkaline phosphatase (ALP). Iron dose-dependently attenuated ECM mineralization and decreased the expressions of Runx2 and OCN. Iron accomplished complete inhibition of osteogenic differentiation of BMSCs at 50µmol/L concentration. We demonstrated that in response to iron BMSCs upregulated the expression of ferritin. Administration of exogenous ferritin mimicked the anti-osteogenic effect of iron, and blocked the upregulation of Runx2, OCN and ALP. Iron overload in mice was associated with elevated ferritin and decreased Runx2 mRNA levels in compact bone osteoprogenitor cells. The inhibitory effect of iron is specific toward osteogenic differentiation of MSCs as neither chondrogenesis nor adipogenesis were influenced by excess iron. We concluded that iron and ferritin specifically inhibit osteogenic commitment and differentiation of BMSCs both in vitro and in vivo.
Asunto(s)
Ferritinas/biosíntesis , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Osteogénesis/fisiología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/fisiología , Calcio/metabolismo , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ferritinas/farmacología , Humanos , Hierro/administración & dosificación , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fosfatos/metabolismo , Fosfatos/farmacologíaRESUMEN
Calcification of the human lens has been described in senile cataracts and in young patients with congenital cataract or chronic uveitis. Lens calcification is also a major complication of cataract surgery and plays a role in the opacification of intraocular lenses. A cell-mediated process has been suggested in the background of lens calcification, but so far the exact mechanism remained unexplored. Lens calcification shares remarkable similarities with vascular calcification; in both pathological processes hydroxyapatite accumulates in the soft tissue. Vascular calcification is a regulated, cell-mediated process in which vascular cells undergo osteogenic differentiation. Our objective was to investigate whether human lens epithelial cells (HuLECs) can undergo osteogenic transition in vitro, and whether this process contributes to lens calcification. We used inorganic phosphate (Pi) and Ca to stimulate osteogenic differentiation of HuLECs. Osteogenic stimuli (2.5mmol/L Pi and 1.2mmol/L Ca) induced extracellular matrix mineralization and Ca deposition in HuLECs with the critical involvement of active Pi uptake. Osteogenic stimuli almost doubled mRNA expressions of osteo-/chondrogenic transcription factors Runx2 and Sox9, which was accompanied by a 1.9-fold increase in Runx2 and a 5.5-fold increase in Sox9 protein expressions. Osteogenic stimuli induced mRNA and protein expressions of alkaline phosphatase and osteocalcin in HuLEC. Ca content was higher in human cataractous lenses, compared to non-cataractous controls (n=10). Osteocalcin, an osteoblast-specific protein, was expressed in 2 out of 10 cataractous lenses. We conclude that osteogenic stimuli induce osteogenic differentiation of HuLECs and propose that this mechanism might play a role in lens calcification.
Asunto(s)
Calcinosis/patología , Cristalino/patología , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Calcinosis/etiología , Calcinosis/metabolismo , Calcio/metabolismo , Catarata/etiología , Catarata/metabolismo , Catarata/patología , Diferenciación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Femenino , Humanos , Cristalino/metabolismo , Masculino , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis , Fosfatos/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Regulación hacia ArribaRESUMEN
Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using ß-glycerophosphate with activated vitamin D3 , or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast-like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H-1,2-Dithiole-3-thione was able to inhibit the SMC transition into osteoblast-like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.
Asunto(s)
Ferritinas/metabolismo , Miocitos del Músculo Liso/fisiología , Osteoblastos/fisiología , Fosfatasa Alcalina/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Calcitriol/metabolismo , Calcio/metabolismo , Células Cultivadas , Colecalciferol/metabolismo , Ergocalciferoles/metabolismo , Glicerofosfatos/farmacología , Humanos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Fosfatos/metabolismo , Tionas/farmacología , Tiofenos/farmacología , Calcificación Vascular/metabolismo , Calcificación Vascular/fisiopatologíaRESUMEN
Introduction: Vascular calcification is accelerated in patients with chronic kidney disease (CKD) and increases the risk of cardiovascular events. CKD is frequently associated with anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor for the treatment of CKD-associated anemia that enhances erythropoiesis through the activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Studies showed that DPD promotes osteogenic differentiation of human aortic smooth muscle cells (HAoSMCs) and increases aorta calcification in mice with CKD. HIF-1 activation has been linked with endoplasmic reticulum (ER) stress; therefore, here we investigated the potential contribution of ER stress, particularly activating transcription factor 4 (ATF4), to the pro-calcification effect of DPD. Methods: Here, we used an adenine-induced CKD mouse model and HAoSMCs as an in vitro vascular calcification model to study the effect of DPD. Results: DPD treatment (15 mg/kg/day) corrects anemia but increases the expression of hypoxia (Glut1, VEGFA), ER stress (ATF4, CHOP, and GRP78), and osteo-/chondrogenic (Runx2, Sox9, BMP2, and Msx2) markers and accelerates aorta and kidney calcification in CKD mice. DPD activates the PERK/eIF2α/ATF4/CHOP pathway and promotes high phosphate-induced osteo-/chondrogenic differentiation of HAoSMCs. Inhibition of ER stress with 4-PBA or silencing of ATF4 attenuates HAoSMC calcification. DPD-induced ATF4 expression is abolished in the absence of HIF-1α; however, knockdown of ATF4 does not affect HIF-1α expression. Conclusion: We concluded that DPD induces ER stress in vitro and in vivo, in which ATF4 serves as a downstream effector of HIF-1 activation. Targeting ATF4 could be a potential therapeutic approach to attenuate the pro-calcific effect of DPD.
RESUMEN
Patients with advanced chronic kidney disease (CKD) have elevated circulating calcium × phosphate product levels and exhibit soft tissue calcification. Besides the cardiovascular system, calcification is commonly observed in the cornea in CKD patients on hemodialysis. Cardiovascular calcification is a cell-mediated, highly regulated process, and we hypothesized that a similar regulatory mechanism is implicated in corneal calcification with the involvement of corneal epithelial cells (CECs). We established a mouse model of CKD-associated corneal calcification by inducing CKD in DBA/2J mice with an adenine and high phosphate diet. CKD was associated with aorta and corneal calcification as detected by OsteoSense staining and corneal Ca measurement (1.67-fold elevation, p < 0.001). In vitro, excess phosphate and Ca induced human CEC calcification in a dose-dependent and synergistic manner, without any influence on cell viability. High phosphate and Ca-containing osteogenic medium (OM; 2.5 mmol/L excess phosphate and 0.6 mmol/L excess Ca over control) increased the protein expression of Runx2 and induced its nuclear translocation. OM increased the expression of the bone-specific Ca-binding protein osteocalcin (130-fold increase, p < 0.001). Silencing of Runx2 attenuated OM-induced CEC calcification. Immunohistology revealed upregulation of Runx2 and overlapping between the Runx2 and the Alizarin red positive areas of calcification in the cornea of CKD mice. This work sheds light on the mechanism of CKD-induced corneal calcification and provides tools to test calcification inhibitors for the prevention of this detrimental process.
Asunto(s)
Calcinosis , Calcio , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Osteoblastos , Fosfatos , Insuficiencia Renal Crónica , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/complicaciones , Ratones , Humanos , Osteoblastos/metabolismo , Osteoblastos/patología , Fosfatos/metabolismo , Calcio/metabolismo , Calcinosis/patología , Calcinosis/metabolismo , Epitelio Corneal/patología , Epitelio Corneal/metabolismo , Masculino , Ratones Endogámicos DBA , Células Epiteliales/metabolismo , Células Epiteliales/patología , Modelos Animales de Enfermedad , FenotipoRESUMEN
Introduction: Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Methods and result: Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model. Discussion: HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.
RESUMEN
The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large-scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 µM patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 µM) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 µM) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects.
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Cromatina/efectos de los fármacos , Micotoxinas/farmacología , Patulina/farmacología , Schizosaccharomyces/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Cromatina/genética , Necrosis/genética , Necrosis/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Cataract, an opacification in the crystalline lens, is a leading cause of blindness. Deposition of hydroxyapatite occurs in a cataractous lens that could be the consequence of osteogenic differentiation of lens epithelial cells (LECs). Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the transcription of a wide range of cytoprotective genes. Nrf2 upregulation attenuates cataract formation. Here we aimed to investigate the effect of Nrf2 system upregulation in LECs calcification. We induced osteogenic differentiation of human LECs (HuLECs) with increased phosphate and calcium-containing osteogenic medium (OM). OM-induced calcium and osteocalcin deposition in HuLECs. We used heme to activate Nrf2, which strongly upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1). Heme-mediated Nrf2 activation was dependent on the production of reactive oxygens species. Heme inhibited Ca deposition, and the OM-induced increase of osteogenic markers, RUNX2, alkaline phosphatase, and OCN. Anti-calcification effect of heme was lost when the transcriptional activity of Nrf2 or the enzyme activity of HO-1 was blocked with pharmacological inhibitors. Among products of HO-1 catalyzed heme degradation iron mimicked the anti-calcification effect of heme. We concluded that heme-induced upregulation of the Nrf2/HO-1 system inhibits HuLECs calcification through the liberation of heme iron.
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Aims: Chronic kidney disease (CKD) is frequently associated with other chronic diseases including anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor, a member of a family of those new generation drugs that increase erythropoiesis via activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Previous studies showed that HIF-1 activation is ultimately linked to acceleration of vascular calcification. We aimed to investigate the effect of DPD on high phosphate-induced calcification. Methods and Results: We investigated the effect of DPD on calcification in primary human aortic vascular smooth muscle cells (VSMCs), in mouse aorta rings, and an adenine and high phosphate-induced CKD murine model. DPD stabilized HIF-1α and HIF-2α and activated the HIF-1 pathway in VSMCs. Treatment with DPD increased phosphate-induced calcification in cultured VSMCs and murine aorta rings. Oral administration of DPD to adenine and high phosphate-induced CKD mice corrected anemia but increased aortic calcification as assessed by osteosense staining. The inhibition of the transcriptional activity of HIF-1 by chetomin or silencing of HIF-1α attenuated the effect of DPD on VSMC calcification. Conclusion: Clinical studies with a long follow-up period are needed to evaluate the possible risk of sustained activation of HIF-1 by DPD in accelerating medial calcification in CKD patients with hyperphosphatemia.
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Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.
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Natterin is an aerolysin-like pore-forming toxin responsible for the toxic effects of the venom of the medically significant fish Thalassophryne nattereri. Using a combination of pharmacologic and genetic loss-of-function approaches we conduct a systematic investigation of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model. Our data confirmed the capacity of Natterin to induce a strong and sustained neutrophilic inflammation leading to systemic inflammatory lung infiltration and revealed overlapping regulatory paths in its control. We found that Natterin induced the extracellular release of mature IL-1ß and the sustained production of IL-33 by bronchial epithelial cells. We confirmed the dependence of both ST2/IL-33 and IL-17A/IL-17RA signaling on the local and systemic neutrophils migration, as well as the crucial role of IL-1α, caspase-1 and caspase-11 for neutrophilic inflammation. The inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis. Finally, neutrophilic inflammation was mediated by non-canonical NLRP6 and NLRC4 adaptors through ASC interaction, independent of NLRP3. Our data highlight that the inflammatory process dependent on non-canonical inflammasome activation can be a target for pharmacological intervention in accidents by T. nattereri, which does not have adequate specific therapy.
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Caspasa 1/metabolismo , Caspasas Iniciadoras/metabolismo , Venenos de los Peces/farmacología , Inflamasomas/metabolismo , Inflamación/inmunología , Interleucina-1beta/metabolismo , Pulmón/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Peritonitis/inducido químicamente , Receptores de Superficie Celular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/genética , Caspasas Iniciadoras/genética , Femenino , Inflamasomas/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/enzimología , Pulmón/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Peritonitis/enzimología , Peritonitis/genética , Peritonitis/inmunología , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Transducción de SeñalRESUMEN
Vascular calcification is the deposition of hydroxyapatite crystals in the medial or intimal layers of arteries that is usually associated with other pathological conditions including but not limited to chronic kidney disease, atherosclerosis and diabetes. Calcification is an active, cell-regulated process involving the phenotype transition of vascular smooth muscle cells (VSMCs) from contractile to osteoblast/chondrocyte-like cells. Diverse triggers and signal transduction pathways have been identified behind vascular calcification. In this review, we focus on the role of reactive oxygen species (ROS) in the osteochondrogenic phenotype switch of VSMCs and subsequent calcification. Vascular calcification is associated with elevated ROS production. Excessive ROS contribute to the activation of certain osteochondrogenic signal transduction pathways, thereby accelerating osteochondrogenic transdifferentiation of VSMCs. Inhibition of ROS production and ROS scavengers and activation of endogenous protective mechanisms are promising therapeutic approaches in the prevention of osteochondrogenic transdifferentiation of VSMCs and subsequent vascular calcification. The present review discusses the formation and actions of excess ROS in different experimental models of calcification, and the potential of ROS-lowering strategies in the prevention of this deleterious condition.
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Hemolytic diseases are characterized by an accelerated breakdown of red blood cells (RBCs) and the release of hemoglobin (Hb). Following, RBC lysis Hb oxidation occurs with the formation of different redox states of Hb (metHb and ferrylHb) and the release of heme. ferrylHb is unstable and decomposes to metHb with the concomitant formation of globin radicals and eventually covalently crosslinked Hb multimers. The goal of the present study was to determine the concentrations of the different redox states of Hb in biological samples during hemolytic conditions. We used plasma and urine samples of mice with intravascular hemolysis and human cerebrospinal fluid (CSF) samples following intraventricular hemorrhage. Because ferrylHb is highly unstable, we also addressed the fate of this species. metHb and free heme time-dependently accumulate in plasma and CSF samples following intravascular hemolysis and intraventricular hemorrhage, respectively. ferrylHb is hardly detectable in the biological samples during hemolytic conditions. Under in vitro conditions, ferrylHb decomposes quickly to metHb, which process is associated with the formation of covalently crosslinked Hb multimers. We detected these covalently crosslinked Hb multimers in plasma, urine, and CSF samples during hemolytic conditions. Because globin modification is specific for these Hb forms, we propose to call this heterogeneous form of Hb produced during ferrylHb decomposition as globin-modified oxidized Hb (gmoxHb). Understanding the formation and the contribution of gmoxHb species to the pathogenesis of hemolytic conditions could have therapeutic implications in the treatment of hemolytic diseases.
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Análisis Químico de la Sangre/métodos , Eritrocitos/metabolismo , Hemoglobinas/química , Animales , Sangre , Humanos , RatonesRESUMEN
Intraventricular hemorrhage (IVH) is a frequent complication of prematurity that is associated with high neonatal mortality and morbidity. IVH is accompanied by red blood cell (RBC) lysis, hemoglobin (Hb) oxidation, and sterile inflammation. Here we investigated whether extracellular Hb, metHb, ferrylHb, and heme contribute to the inflammatory response after IVH. We collected cerebrospinal fluid (CSF) (n = 20) from premature infants with grade III IVH at different time points after the onset of IVH. Levels of Hb, metHb, total heme, and free heme were the highest in CSF samples obtained between days 0 and 20 after the onset of IVH and were mostly non-detectable in CSF collected between days 41 and 60 of post-IVH. Besides Hb monomers, we detected cross-linked Hb dimers and tetramers in post-IVH CSF samples obtained in days 0-20 and 21-40, but only Hb tetramers were present in CSF samples obtained after 41-60 days. Vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) levels were higher in CSF samples obtained between days 0 and 20 than in CSF collected between days 41 and 60 of post-IVH. Concentrations of VCAM-1, intercellular adhesion molecule-1 (ICAM-1), and IL-8 strongly correlated with total heme levels in CSF. Applying the identified heme sources on human brain microvascular endothelial cells revealed that Hb oxidation products and free heme contribute to the inflammatory response. We concluded that RBC lysis, Hb oxidation, and heme release are important components of the inflammatory response in IVH. Pharmacological interventions targeting cell-free Hb, Hb oxidation products, and free heme could have potential to limit the neuroinflammatory response following IVH.