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1.
Calcif Tissue Int ; 115(1): 85-96, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38733412

RESUMEN

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl-/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable 'post hoc loading' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases.


Asunto(s)
Alelos , Canales de Cloruro , Nanopartículas , Osteopetrosis , Fenotipo , ARN Interferente Pequeño , Animales , Canales de Cloruro/genética , Osteopetrosis/genética , Osteopetrosis/terapia , Ratones , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Huesos/metabolismo , Huesos/efectos de los fármacos , Modelos Animales de Enfermedad
2.
Front Pharmacol ; 14: 1151144, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37153788

RESUMEN

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

3.
Front Physiol ; 13: 1059369, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36467702

RESUMEN

The Calcium-Sensing Receptor (CaSR) is a member of the class C of G-proteins coupled receptors (GPCRs), it plays a pivotal role in calcium homeostasis by directly controlling calcium excretion in the kidneys and indirectly by regulating parathyroid hormone (PTH) release from the parathyroid glands. The CaSR is found to be ubiquitously expressed in the body, playing a plethora of additional functions spanning from fluid secretion, insulin release, neuronal development, vessel tone to cell proliferation and apoptosis, to name but a few. The present review aims to elucidate and clarify the emerging regulatory effects that the CaSR plays in inflammation in several tissues, where it mostly promotes pro-inflammatory responses, with the exception of the large intestine, where contradictory roles have been recently reported. The CaSR has been found to be expressed even in immune cells, where it stimulates immune response and chemokinesis. On the other hand, CaSR expression seems to be boosted under inflammatory stimulus, in particular, by pro-inflammatory cytokines. Because of this, the CaSR has been addressed as a key factor responsible for hypocalcemia and low levels of PTH that are commonly found in critically ill patients under sepsis or after burn injury. Moreover, the CaSR has been found to be implicated in autoimmune-hypoparathyroidism, recently found also in patients treated with immune-checkpoint inhibitors. Given the tight bound between the CaSR, calcium and vitamin D metabolism, we also speculate about their roles in the pathogenesis of severe acute respiratory syndrome coronavirus-19 (SARS-COVID-19) infection and their impact on patients' prognosis. We will further explore the therapeutic potential of pharmacological targeting of the CaSR for the treatment and management of aberrant inflammatory responses.

4.
Front Endocrinol (Lausanne) ; 13: 1085315, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36714597

RESUMEN

Hematopoietic stem cell transplantation (HSCT) is an established therapeutic strategy for the treatment of malignant (leukemia and lymphoma) and non-malignant (thalassemia, anemia, and immunodeficiency) hematopoietic diseases. Thanks to the improvement in patient care and the development of more tolerable conditioning treatments, which has extended the applicability of therapy to the elderly, a growing number of patients have successfully benefited from HSCT therapy and, more importantly, HSCT transplant-related mortality has consistently reduced in recent years. However, concomitantly to long term patient survival, a growing incidence of late HSCT-related sequelae has been reported, being variably associated with negative effects on quality of life of patients and having a non-negligible impact on healthcare systems. The most predominantly observed HSCT-caused complications are chronic alterations of the endocrine system and metabolism, which endanger post-operative quality of life and increase morbidity and mortality of transplanted patients. Here, we specifically review the current knowledge on HSCT-derived side-effects on the perturbation of mineral metabolism; in particular, the homeostasis of calcium, focusing on current reports regarding osteoporosis and recurrent renal dysfunctions that have been observed in a percentage of HSC-transplanted patients. Possible secondary implications of conditioning treatments for HSCT on the physiology of the parathyroid glands and calcium homeostasis, alone or in association with HSCT-caused renal and bone defects, are critically discussed as well.


Asunto(s)
Calcio , Trasplante de Células Madre Hematopoyéticas , Humanos , Anciano , Calidad de Vida , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Progresión de la Enfermedad , Homeostasis , Sistema Endocrino , Minerales
5.
Int J Mol Sci ; 22(18)2021 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-34576291

RESUMEN

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on ß-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.


Asunto(s)
Neoplasias del Colon/metabolismo , Espacio Extracelular/metabolismo , Receptores Sensibles al Calcio/química , Receptores Sensibles al Calcio/metabolismo , Neoplasias del Colon/patología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HT29 , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Modelos Moleculares , Conformación Molecular , Receptores Sensibles al Calcio/genética , Estereoisomerismo
6.
Biochim Biophys Acta Mol Cell Res ; 1867(12): 118836, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32861746

RESUMEN

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 µM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.


Asunto(s)
Calcimiméticos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Receptores Sensibles al Calcio/genética , Receptores Acoplados a Proteínas G/genética , Animales , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Subunidad p19 de la Interleucina-23/genética , Interleucina-8/genética , Ratones , Fenetilaminas/farmacología , Propilaminas/farmacología
7.
Nutrients ; 11(12)2019 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-31888253

RESUMEN

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect-it shortened the colons (5.3 ± 0.1 cm vs. 6.1 ± 0.2 cm normal diet, p < 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-γ, (4.2-fold, p < 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-α (4.4-fold, p < 0.05) and IL-6 (4.9-fold, p < 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 ± 19.1 vs. 21.9 ± 14.3 area under the curve, p < 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases.


Asunto(s)
Calcio de la Dieta/farmacología , Colitis/metabolismo , Dieta Rica en Proteínas/efectos adversos , Proteínas en la Dieta/farmacología , Receptores Sensibles al Calcio/agonistas , Animales , Colitis/inducido químicamente , Colon/metabolismo , Citocinas/metabolismo , Sulfato de Dextran , Femenino , Inflamación , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos BALB C , Naftalenos/administración & dosificación
8.
World J Gastroenterol ; 24(36): 4119-4131, 2018 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-30271078

RESUMEN

The extracellular calcium-sensing receptor (CaSR) is best known for its action in the parathyroid gland and kidneys where it controls body calcium homeostasis. However, the CaSR has different roles in the gastrointestinal tract, where it is ubiquitously expressed. In the colon, the CaSR is involved in controlling multiple mechanisms, including fluid transport, inflammation, cell proliferation and differentiation. Although the expression pattern and functions of the CaSR in the colonic microenvironment are far from being completely understood, evidence has been accumulating that the CaSR might play a protective role against both colonic inflammation and colorectal cancer. For example, CaSR agonists such as dipeptides have been suggested to reduce colonic inflammation, while dietary calcium was shown to reduce the risk of colorectal cancer. CaSR expression is lost in colonic malignancies, indicating that the CaSR is a biomarker for colonic cancer progression. This dual anti-inflammatory and anti-tumourigenic role of the CaSR makes it especially interesting in colitis-associated colorectal cancer. In this review, we describe the clinical and experimental evidence for the role of the CaSR in colonic inflammation and colorectal cancer, the intracellular signalling pathways which are putatively involved in these actions, and the possibilities to exploit these actions of the CaSR for future therapies of colonic inflammation and cancer.


Asunto(s)
Antineoplásicos/uso terapéutico , Colitis/patología , Neoplasias Colorrectales/patología , Fármacos Gastrointestinales/uso terapéutico , Receptores Sensibles al Calcio/metabolismo , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/patología , Colitis/complicaciones , Colitis/tratamiento farmacológico , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/mortalidad , Progresión de la Enfermedad , Regulación hacia Abajo , Fármacos Gastrointestinales/farmacología , Humanos , Pronóstico , Receptores Sensibles al Calcio/análisis , Receptores Sensibles al Calcio/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos
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