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- Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseasesPublication . Viegas, Carla; Costa, Ruben M.; Santos, Lúcia; Videira, Paula A.; Silva, Zelia; Araujo, Nuna C. P.; Macedo, Anjos L.; Matos, Antonio P.; Vermeer, Cees; Simes, DinaCalcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.
- Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological CalcificationsPublication . Viegas, Carla; Cavaco, Sofia; Neves, Pedro L.; Ferreira, Ana; Joao, Alexandre; Williamson, Matthew K.; Price, Paul A.; Cancela, M. Leonor; Simes, DinaMineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization. (Am J Pathol 2009, 175:2288-2298; DOI; 10.2353/ajpath.2009.090474)
- Gla-Rich protein, magnesium and phosphate associate with mitral and aortic valves calcification in Didabetic patients with moderate CKDPublication . Silva, Ana P.; Viegas, Carla; Guilherme, Patrícia; Tavares, Nelson; Dias, Carolina; Rato, Fátima; Santos, Nélio; Faísca, Marília; de Almeida, Edgar; Neves, Pedro L.; Simes, Dina C.Accelerated and premature cardiovascular calcification is a hallmark of chronic kidney disease (CKD) patients. Valvular calcification (VC) is a critical indicator of cardiovascular disease and all-cause mortality in this population, lacking validated biomarkers for early diagnosis. Gla-rich protein (GRP) is a cardiovascular calcification inhibitor recently associated with vascular calcification, pulse pressure, mineral metabolism markers and kidney function. Here, we examined the association between GRP serum levels and mitral and aortic valves calcification in a cohort of 80 diabetic patients with CKD stages 2–4. Mitral and aortic valves calcification were detected in 36.2% and 34.4% of the patients and associated with lower GRP levels, even after adjustments for age and gender. In this pilot study, univariate, multivariate and Poisson regression analysis, show that low levels of GRP and magnesium (Mg), and high levels of phosphate (P) are associated with mitral and aortic valves calcification. Receiver operating characteristic (ROC) curves showed that the area under the curve (AUC) values of GRP for mitral (0.762) and aortic (0.802) valves calcification were higher than those of Mg and P. These results suggest that low levels of GRP and Mg, and high levels of P, are independent and cumulative risk factors for VC in this population; the GRP diagnostic value might be potentially useful in cardiovascular risk assessment.
- Targeting a silent disease: vascular calcification in chronic Kidney diseasePublication . Marreiros, Catarina; Viegas, Carla; Simes, DinaChronic kidney disease (CKD) patients have a higher risk of developing early cardiovascular disease (CVD). Although vascular calcification (VC) is one of the strongest predictors of CVD risk, its diagnosis among the CKD population remains a serious clinical challenge. This is mainly due to the complexity of VC, which results from various interconnected pathological mechanisms occurring at early stages and at multiples sites, affecting the medial and intimal layers of the vascular tree. Here, we review the most used and recently developed imaging techniques, here referred to as imaging biomarkers, for VC detection and monitoring, while discussing their strengths and limitations considering the specificities of VC in a CKD context. Although imaging biomarkers have a crucial role in the diagnosis of VC, with important insights into CVD risk, circulating biomarkers represent an added value by reflecting the molecular dynamics and mechanisms involved in VC pathophysiological pathways, opening new avenues into the early detection and targeted interventions. We propose that a combined strategy using imaging and circulating biomarkers with a role in multiple VC molecular mechanisms, such as Fetuin-A, Matrix Gla protein, Gla-rich protein and calciprotein particles, should represent high prognostic value for management of CVD risk in the CKD population.
- A dual role for GRP in cardiovascular diseasePublication . S B Viegas, Carla; Simes, DManagement and prevention of cardiovascular disease (CVD) represents one of the major health challenges worldwide. CVD is the leading cause of death globally despite all research efforts on last decades regarding the molecular mechanisms and processes involved on its development and progression. Chronic Kidney Disease (CKD) is an independent risk factor and promotor of CVD events, representing a considerable economic cost for the health system. CVD is the leading cause of death in all CKD stages, accounting for half the number of deaths in this population.
- Gla rich protein (GRP) mediates vascular smooth muscle cell (VSMC) osteogenic differentiation, extracellular vesicle (EV) calcification propensity, and immunomodulatory propertiesPublication . Maia, Teresa M.; Macedo, Anjos L.; Matos, António P.; Neves, José; Viegas, Carla; Camilo Carreira, Joana Sofia; Simes, DinaVascular calcification (VC) is a complex process involving vascular smooth muscle cell (VSMC) osteogenic differentiation, inflammation, and extracellular vesicle (EV) calcification and communication networks. Gla rich protein (GRP) is a calcification inhibitor involved in most of these processes. However, the molecular mechanism of GRP in VC and the specific characteristics, cargo, and functionality of calcifying EVs require further elucidation. Here, we use a combination of human ex vivo aortic fragments and primary vascular smooth muscle cell (VSMC) models to obtain new information on GRP function in VC and EVs released by VSMCs. We demonstrate that GRP inhibits VSMC osteogenic differentiation through downregulation of bone-related proteins and upregulation of mineralization inhibitors, with decreased mineral crystallinity in EVs deposited into the tissue extracellular matrix (ECM). EVs isolated by ultracentrifugation at 30K and 100K from the cell media (CM) and deposited in the ECM from control (CTR) and mineralizing (MM) VSMCs were biochemically, physically, and proteomically characterized. Four different EV populations were identified with shared markers commonly present in all EVs but with unique protein cargo and specific molecular profiles. Comparative proteomics identified several regulated proteins specifically loaded into MM EV populations associated with multiple processes involved in VC. Functional analysis demonstrated that 30K and 100K ECM-MM EVs with higher calcium and lower GRP levels induced macrophage inflammation. Our findings reinforce the functional relevance of GRP in multiple VC processes and suggest that ECM EVs released under calcification stress function as a new signaling axis on the calcification-inflammation cycle.
- Gla-Rich protein is associated with vascular calcification, inflammation, and mineral markers in peritoneal dialysis patientsPublication . MARREIROS, CATARINA; Viegas, Carla; Malho Guedes, Anabela; Silva, Ana Paula de Andrade; Águas, Ana Catarina; Faísca, Marília; Schurgers, Leon; Simes, DinaVascular calcification (VC) is a crucial risk factor for cardiovascular diseases (CVD), particularly in chronic kidney disease (CKD) populations. However, the specific relationship between VC and end-stage renal disease (ESRD) patients undergoing peritoneal dialysis (PD) remains to be fully understood. The identification of new biomarkers to improve VC diagnosis and monitoring would significantly impact cardiovascular risk management in these high-risk patients. Gla-rich protein (GRP) is a VC inhibitor and an anti-inflammatory agent and thus is a potential VC marker in CKD. Here we explored the potential role of GRP as a marker for CVD and investigated the impact of VC in 101 PD patients. Methods: Circulating total Gla-rich protein (tGRP) was quantified in serum and in 24 h dialysate samples. VC score (VCS) was determined using the Adragão method. Results: Serum tGRP was negatively associated with VCS, serum calcium (Ca), phosphate (P), and high-sensitivity C-reactive protein (hsCRP), while it was positively associated with magnesium (Mg). A total of 35.6% of PD patients presented with extensive calcifications (VCS ≥ 3), and the lowest tGRP serum levels were present in this group (419.4 ± 198.5 pg/mL). tGRP in the 24 h dialysate was also negatively associated with VCS and with serum Ca and P. Moreover, serum Ca, P, and VCS were identified as independent determinants of serum tGRP levels. Conclusions: The association of serum tGRP with VC, mineral, and inflammation markers reinforces its potential use as a novel VC biomarker in CKD patients undergoing PD.