Vitamin K as a powerful micronutrient in aging and age-related diseases: pros and cons from clinical studies

Vitamin K is a multifunctional micronutrient implicated in age-related diseases such as cardiovascular diseases, osteoarthritis and osteoporosis. Although vitamin K-dependent proteins (VKDPs) are described to have a crucial role in the pathogenesis of these diseases, novel roles have emerged for vitamin K, independently of its role in VKDPs carboxylation. Vitamin K has been shown to act as an anti-inflammatory by suppressing nuclear factor κB (NF-κB) signal transduction and to exert a protective effect against oxidative stress by blocking the generation of reactive oxygen species. Available clinical evidences indicate that a high vitamin K status can exert a protective role in the inflammatory and mineralization processes associated with the onset and progression of age-related diseases. Also, vitamin K involvement as a protective super-micronutrient in aging and 'inflammaging' is arising, highlighting its future use in clinical practice. In this review we summarize current knowledge regarding clinical data on vitamin K in skeletal and cardiovascular health, and discuss the potential of vitamin K supplementation as a health benefit. We describe the clinical evidence and explore molecular aspects of vitamin K protective role in aging and age-related diseases, and its involvement as a modulator in the interplay between pathological calcification and inflammation processes.Agência financiadora Portuguese Society of Nephrology (SPN) Portuguese national funds from FCT-Foundation for Science and Technology UID/Multi/04326/2019info:eu-repo/semantics/publishedVersio

Amentadione from the Alga Cystoseira usneoides as a Novel Osteoarthritis Protective Agent in an Ex Vivo Co-Culture OA Model

Osteoarthritis (OA) remains a prevalent chronic disease without effective prevention and treatment. Amentadione (YP), a meroditerpenoid purified from the alga Cystoseira usneoides, has demonstrated anti-inflammatory activity. Here, we investigated the YP anti-osteoarthritic potential, by using a novel OA preclinical drug development pipeline designed to evaluate the anti-inflammatory and anti-mineralizing activities of potential OA-protective compounds. The workflow was based on in vitro primary cell cultures followed by human cartilage explants assays and a new OA co-culture model, combining cartilage explants with synoviocytes under interleukin-1 beta (IL-1 beta) or hydroxyapatite (HAP) stimulation. A combination of gene expression analysis and measurement of inflammatory mediators showed that the proposed model mimicked early disease stages, while YP counteracted inflammatory responses by downregulation of COX-2 and IL-6, improved cartilage homeostasis by downregulation of MMP3 and the chondrocytes hypertrophic differentiation factors Col10 and Runx2. Importantly, YP downregulated NF-kappa B gene expression and decreased phosphorylated IkB alpha/total IkB alpha ratio in chondrocytes. These results indicate the co-culture as a relevant pre-clinical OA model, and strongly suggest YP as a cartilage protective factor by inhibiting inflammatory, mineralizing, catabolic and differentiation processes during OA development, through inhibition of NF-kappa B signaling pathways, with high therapeutic potential

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseases

Calcification-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.Portuguese Science and Technology Foundation (FCT) [PTDC/SAU-ORG/117266/2010, PTDC/BIM-MEC/1168/2012, UID/Multi/ 04326/2013]; FCT fellowships [SFRH/BPD/70277/2010, SFRH/BD/111824/2015

Vitamin K as a Powerful Micronutrient in Aging and Age-Related Diseases: Pros and Cons from Clinical Studies

Vitamin K is a multifunctional micronutrient implicated in age-related diseases such as cardiovascular diseases, osteoarthritis and osteoporosis. Although vitamin K-dependent proteins (VKDPs) are described to have a crucial role in the pathogenesis of these diseases, novel roles have emerged for vitamin K, independently of its role in VKDPs carboxylation. Vitamin K has been shown to act as an anti-inflammatory by suppressing nuclear factor κB (NF-κB) signal transduction and to exert a protective effect against oxidative stress by blocking the generation of reactive oxygen species. Available clinical evidences indicate that a high vitamin K status can exert a protective role in the inflammatory and mineralization processes associated with the onset and progression of age-related diseases. Also, vitamin K involvement as a protective super-micronutrient in aging and ‘inflammaging’ is arising, highlighting its future use in clinical practice. In this review we summarize current knowledge regarding clinical data on vitamin K in skeletal and cardiovascular health, and discuss the potential of vitamin K supplementation as a health benefit. We describe the clinical evidence and explore molecular aspects of vitamin K protective role in aging and age-related diseases, and its involvement as a modulator in the interplay between pathological calcification and inflammation processes

Nanoencapsulação como uma nova abordagem de entrega para aplicações terapêuticas de proteína rica em gla (GRP)

Gla rich protein (GRP) is a vitamin K dependent protein, shown to function as an inhibitor of pathological calcification and as an anti-inflammatory agent, with potential therapeutic use for age-related diseases such as osteoarthritis (OA) [1,2]. OA is a leading cause of disability and morbidity in the older population and constitutes a major world wide challenge for our health system. Presently, there are no drugs approved that can prevent, stop, or even restrain progression of OA. GRP has been shown to be able to lower inflammation and mineralisation processes in the articular tissue. Chitosan/tripolyphosphate (TPP) nanoparticles were selected for this study due to their biocompatibility, biodegradability and capacity to overcome the problem of low solubility of GRP in physiological conditions. This study aims to produce and characterise chitosan/TPP nanoparticles as GRP-delivery vehicles and test its anti-inflammatory potential in human macrophages.info:eu-repo/semantics/publishedVersio

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation

LA/P/0101/2020 LA/P/0140/2020 AAC nº 41/ALG/2020—Project nº 072583—NUTRISAFEChronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavail-ability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.publishersversionpublishe

Overexpression of GRP in THP-1 cells rescues hydroxyapatite induced inflammation.

<p>THP-1 cells were transiently transfected with GRP mRNA for 24 h and either maintained in control conditions (C, T) or stimulated with HA (C-HA, T-HA) for additional 48 h. Gene expression of GRP (A) and the inflammatory marker genes NFkB (B) and TNFα (C) were determined by qPCR (A), and levels of TNFα accumulation were measured by ELISA in conditioned culture media (D). (A-C) Data are presented as means (n = 6) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P< = 0.01 (**), P< = 0.001 (***) and P<0.0001 (****). <i>ns</i>, non-significant. (D) Data are presented as means (n = 4) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.001 (***) and P<0.0001 (****).</p

GRP and MGP are produced by leukocytes as γ-carboxylated proteins.

<p>Leukocyte subsets neutrophils (Neut), monocytes (Mon), and T lymphocytes (T-Lym) were isolated from human buffy coats, and macrophages (Mac) and dendritic cells (DC) were obtained by differentiation of isolated monocytes in culture. After purity assessment by flow cytometry, these isolated leukocyte populations were used to evaluate GRP and MGP production at gene and protein levels. (A) Relative fold expression (arbitrary units) of GRP and MGP determined by qPCR in isolated leukocytes. Means are presented as replicates of 3 biological healthy donors using 18S as housekeeping gene. (B) Total protein extracts of the isolated leukocyte populations were obtained with RIPA buffer and quantified using the Micro BCA kit. Five μg, ten μg and fifteen μg of total protein extracts from T Lym, Mon and Mac, respectively, were analyzed by Western blot to detect total GRP (tGRP) and total MGP (tMGP) protein forms using the validated CTerm-GRP and tMGP antibodies, and γ-carboxylated GRP (cGRP) and MGP (cMGP) protein forms using conformation-specific antibodies. Position of relevant molecular mass markers (kDa) is indicated on the left side. (C) Qualitative gene expression analysis of the γ-carboxylated related enzymes VKOR and GGCX by RT-PCR in T lymphocytes, monocyte and neutrophils. 18S amplification was used as loading control for sample integrity.</p

γ-carboxylated GRP and MGP are produced in THP-1 cell line.

<p>THP-1 and THP-1 MoM differentiated with 25 ng/ml of PMA during 48h were cultured in control conditions and harvested for RNA and protein extraction. (A) Qualitative gene expression analysis of GRP, MGP, VKOR and GGCX by RT-PCR in undifferentiated THP-1 representing monocyte cells (THP-1), and in differentiated THP-1 representing macrophages (THP-1 MoM). (B) Western blot analysis of thirty μg of total RIPA protein extracts of THP-1 and THP-1 MoM cells using the conformation-specific antibodies recognizing γ-carboxylated GRP (cGRP) and MGP (cMGP). Position of relevant molecular mass markers (kDa) is indicated on the right side.</p

GRP reduces TNFα and PGE2 production in THP-1 MoM cells stimulated with LPS.

<p>(A) Differentiated THP-1 MoM cells were treated with 0.5 μg/ml, 0.75 μg/ml and 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h, followed by exposure to 50 ng/ml LPS for additional 24 h. Cells treated with 2 μM dexamethasone (DXM) were used as a positive anti-inflammatory control, and non-stimulated cells (C) as controls to LPS stimulation. Conditioned cell culture media were collected and used to determine TNFα accumulation by ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.05 (*), P< = 0.01 (**), and P< = 0.001 (***). (B) Differentiated THP-1 MoM cells were treated with 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h and exposed to LPS as described in (A), and PGE2 accumulation was determined in conditioned media through ELISA assays. Data are presented as means (n = 2) ± standard error of replicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P< = 0.001 (***) and P<0.0001 (****).</p