Biological Role of Vitamin K-With Particular Emphasis on Cardiovascular and Renal Aspects.

Vitamin K (VK) plays many important functions in the body. The most important of them include the contribution in calcium homeostasis and anticoagulation. Vascular calcification (VC) is one of the most important mechanisms of renal pathology. The most potent inhibitor of this process-matrix Gla protein (MGP) is VK-dependent. Chronic kidney disease (CKD) patients, both non-dialysed and hemodialysed, often have VK deficiency. Elevated uncarboxylated matrix Gla protein (ucMGP) levels indirectly reflected VK deficiency and are associated with a higher risk of cardiovascular events in these patients. It has been suggested that VK intake may reduce the VC and related cardiovascular risk. Vitamin K intake has been suggested to reduce VC and the associated cardiovascular risk. The role and possibility of VK supplementation as well as the impact of anticoagulation therapy on VK deficiency in CKD patients is discussed.

The Role of DMP1 in CKD-MBD.

PURPOSE OF REVIEW: Chronic kidney disease-mineral and bone disorder (CKD-MBD) has become a global health crisis with very limited therapeutic options. Dentin matrix protein 1 (DMP1) is a matrix extracellular protein secreted by osteocytes that has generated recent interest for its possible involvement in CKD-MBD pathogenesis. This is a review of DMP1 established regulation and function, and early studies implicating DMP1 in CKD-MBD. RECENT FINDINGS: Patients and mice with CKD show perturbations of DMP1 expression in bone, associated with impaired osteocyte maturation, mineralization, and increased fibroblast growth factor 23 (FGF23) production. In humans with CKD, low circulating DMP1 levels are independently associated with increased cardiovascular events. We recently showed that DMP1 supplementation lowers circulating FGF23 levels and improves bone mineralization and cardiac outcomes in mice with CKD. Mortality rates are extremely high among patients with CKD and have only marginally improved over decades. Bone disease and FGF23 excess contribute to mortality in CKD by increasing the risk of bone fractures and cardiovascular disease, respectively. Previous studies focused on DMP1 loss-of-function mutations have established its role in the regulation of FGF23 and bone mineralization. Recent studies show that DMP1 supplementation may fill a crucial therapeutic gap by improving bone and cardiac health in CKD.

Bone and heart health in chronic kidney disease: role of dentin matrix protein 1.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) is a condition associated with bone disease and fibroblast growth factor 23 (FGF23) excess that contributes to cardiovascular mortality. Dentin matrix protein 1 (DMP1) is an established regulator of bone mineralization and FGF23 production in osteocytes. To date, DMP1 function has mainly been studied in the context of hereditary hypophosphatemic rickets diseases. This review describes the role of DMP1 as a potential strong candidate to prevent bone disorders, FGF23 elevation and associated cardiac outcomes in CKD. RECENT FINDINGS: Patients and mice with CKD show impaired osteocyte maturation and impaired regulation of DMP1 and FGF23 in bone. New data suggest that impaired DMP1 production contributes to CKD-associated bone and mineral metabolism disorders and we show that DMP1 repletion improves osteocyte alterations, bone mineralization and partially prevents FGF23 elevation. As a result, mice with CKD show attenuated left ventricular hypertrophy and improved survival. SUMMARY: There is an urgent need for new therapeutic strategies to improve bone quality and to lower FGF23 levels in CKD. By preventing osteocyte apoptosis and inhibiting Fgf23 transcription, DMP1 supplementation may represent an ideal approach to improve CKD-associated bone and cardiac outcomes.

Cardiac Extracellular Matrix Modification as a Therapeutic Approach.

The cardiac extracellular matrix (cECM) is comprised of proteins and polysaccharides secreted by cardiac cell types, which provide structural and biochemical support to cardiovascular tissue. The roles of cECM proteins and the associated family of cell surface receptor, integrins, have been explored in vivo via the generation of knockout experimental animal models. However, the complexity of tissues makes it difficult to isolate the effects of individual cECM proteins on a particular cell process or disease state. The desire to further dissect the role of cECM has led to the development of a variety of in vitro model systems, which are now being used not only for basic studies but also for testing drug efficacy and toxicity and for generating therapeutic scaffolds. These systems began with 2D coatings of cECM derived from tissue and have developed to include recombinant ECM proteins, ECM fragments, and ECM mimics. Most recently 3D model systems have emerged, made possible by several developing technologies including, and most notably, 3D bioprinting. This chapter will attempt to track the evolution of our understanding of the relationship between cECM and cell behavior from in vivo model to in vitro control systems. We end the chapter with a summary of how basic studies such as these have informed the use of cECM as a direct therapy.

Expression analysis and functional characterization of the mouse cysteine-rich with EGF-like domains 2.

We have previously identified a novel endoplasmic reticulum (ER) stress-inducible protein, namely, cysteine-rich with EGF-like domains 2 (CRELD2), which is predominantly regulated by ATF6. However, few studies on intrinsic CRELD2 have been published. In the present study, we elucidated the expression of intrinsic CRELD2 in mouse tissues and ER stress- treated Neuro2a cells. Among nine tissues we tested, CRELD2 protein in the heart and skeletal muscles was negligible. CRELD2 expression in Neuro2a cells was induced at the late phase after treatment with tunicamycin (Tm) compared with rapid induction of growth arrest and DNA damage inducible gene 153 (GADD153). On the other hand, another ER stress inducer, thapsigargin, increased the intrinsic CRELD2 secretion from Neuro2a cells. We furthermore established CRELD2-deficient Neuro2a cells to evaluate their features. In combination with the NanoLuc complementary reporter system, which was designed to detect protein-protein interaction in living cells, CRELD2 interacted with not only CRELD2 itself but also with ER localizing proteins in Neuro2a cells. Finally, we investigated the responsiveness of CRELD2-deficient cells against Tm-treatment and found that CRELD2 deficiency did not affect the expression of genes triggered by three canonical ER stress sensors but rendered Neuro2a cells vulnerable to Tm-stimulation. Taken together, these findings provide the novel molecular features of CRELD2, and its further characterization would give new insights into understanding the ER homeostasis and ER stress-induced cellular dysfunctions.

Vitamin K: from coagulation to calcification.

Vitamin K is not only essential for the synthesis of coagulation factors in the liver, but it also strengthens the bones and prevents calcification of the arteries. These effects are mediated through the same mechanism, i.e. carboxylation of Gla target proteins. The discovery of novel Gla proteins that are not associated with blood coagulation or calcium metabolism indicates that vitamin K has additional effects in the pancreas and the central nervous system, for example. As dietary supplements, vitamin K1 of plant origin and vitamins K2 of bacterial origin may exert different effects.

The role of lamina cribrosa cells in optic nerve head fibrosis in glaucoma.

Glaucoma is a chronic progressive optic neuropathy. There are extracellular matrix (ECM) changes associated with optic disc cupping in the optic nerve head (ONH) and subsequent visual field defects. The primary risk factor for onset and progression of glaucoma is raised intraocular pressure (IOP). Elevated IOP causes deformation at the ONH specifically at the lamina cribrosa (LC) region where there is also deposition of ECM causing the LC to initially undergo thickening and posterior migration with eventual shearing and collapse of the LC plates leading to a thin fibrotic connective tissue structure/scar. Cells that populate the LC region of the ONH are those cells that are positive for GFAP (the astrocytes) and those negative for GFAP (the LC cells). The LC cell plays an integral role in ECM remodelling producing ECM when exposed to high level mechanical stretch, TGF- ß1 and a hypoxic environment.

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

[Cardiovascular calcification inhibitors]. / Inhibiteurs des calcifications cardiovasculaires.

Vascular calcification is a marker of cardiovascular risk increase. Age and specific disease such as diabetes or chronic kidney disease are important factors for calcification genesis. Vascular calcification process is a complex phenomenon, involving several activators and inhibitors factors. Indeed, recent works related to in vitro and in vivo experimental studies have led to a better understanding of calcification process and identification of molecules able to modulate this system. This revue will summarize some of these molecules with a particular interest of those with therapeutic relevance. We will present: i) calcium sensing receptor and its modulation by cinacalcet; ii) pyrophosphate supplementation; iii) fetuin A and overall propensity serum test for calcification and finally; iv) matrix-Gla-protein and the use of vitamin K to prevent vascular calcification progression.

Vitamin K1 to slow vascular calcification in haemodialysis patients (VitaVasK trial): a rationale and study protocol.

BACKGROUND: Patients on haemodialysis (HD) exhibit increased cardiovascular mortality associated with accelerated vascular calcification (VC). VC is influenced by inhibitors such as matrix Gla protein (MGP), a protein activated in the presence of vitamin K. HD patients exhibit marked vitamin K deficiency, and supplementation with vitamin K reduces inactive MGP levels in these patients. The VitaVasK trial analyses whether vitamin K1 supplementation affects the progression of coronary and aortic calcification in HD patients. METHODS: VitaVasK is a prospective, randomized, parallel group, multicentre trial (EudraCT No.: 2010-021264-14) that will include 348 HD patients in an open-label, two-arm design. After baseline multi-slice computed tomography (MSCT) of the heart and thoracic aorta, patients with a coronary calcification volume score of at least 100 will be randomized to continue on standard care or to receive additional supplementation with 5 mg vitamin K1 orally thrice weekly. Treatment duration will be 18 months, and MSCT scans will be repeated after 12 and 18 months. Primary end points are the progression of thoracic aortic and coronary artery calcification (calculated as absolute changes in the volume scores at the 18-month MSCT versus the baseline MSCT). Secondary end points comprise changes in Agatston score, mitral and aortic valve calcification as well as major adverse cardiovascular events (MACE) and all-cause mortality. VitaVask also aims to record MACE and all-cause mortality in the follow-up period at 3 and 5 years after treatment initiation. This trial may lead to the identification of an inexpensive and safe treatment or prophylaxis of VC in HD patients.

Vitamin K status and vascular calcification: evidence from observational and clinical studies.

Vascular calcification occurs when calcium accumulates in the intima (associated with atherosclerosis) and/or media layers of the vessel wall. Coronary artery calcification (CAC) reflects the calcium burden within the intima and media of the coronary arteries. In population-based studies, CAC independently predicts cardiovascular disease (CVD) and mortality. A preventive role for vitamin K in vascular calcification has been proposed based on its role in activating matrix Gla protein (MGP), a calcification inhibitor that is expressed in vascular tissue. Although animal and in vitro data support this role of vitamin K, overall data from human studies are inconsistent. The majority of population-based studies have relied on vitamin K intake to measure status. Phylloquinone is the primary dietary form of vitamin K and available supplementation trials, albeit limited, suggest phylloquinone supplementation is relevant to CAC. Yet observational studies have found higher dietary menaquinone, but not phylloquinone, to be associated with less calcification. Vascular calcification is highly prevalent in certain patient populations, especially in those with chronic kidney disease (CKD), and it is plausible vitamin K may contribute to reducing vascular calcification in patients at higher risk. Subclinical vitamin K deficiency has been reported in CKD patients, but studies linking vitamin K status to calcification outcomes in CKD are needed to clarify whether or not improving vitamin K status is associated with improved vascular health in CKD. This review summarizes the available evidence of vitamin K and vascular calcification in population-based studies and clinic-based studies, with a specific focus on CKD patients.

Dentin sialophosphoprotein and dentin matrix protein-1: Two highly phosphorylated proteins in mineralized tissues.

Dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) are highly phosphorylated proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and are essential for proper development of hard tissues such as teeth and bones. In order to understand how they contribute to tissue organization, DSPP and DMP-1 have been analyzed for over a decade using both in vivo and in vitro techniques. Among the five SIBLINGs, the DSPP and DMP-1 genes are located next to each other and their gene and protein structures are most similar. In this review we examine the phenotypes of the genetically engineered mouse models of DSPP and DMP-1 and also introduce complementary in vitro studies into the molecular mechanisms underlying these phenotypes. DSPP affects the mineralization of dentin more profoundly than DMP-1. In contrast, DMP-1 significantly affects bone mineralization and importantly controls serum phosphate levels by regulating serum FGF-23 levels, whereas DSPP does not show any systemic effects. DMP-1 activates integrin signalling and is endocytosed into the cytoplasm whereupon it is translocated to the nucleus. In contrast, DSPP only activates integrin-dependent signalling. Thus it is now clear that both DSPP and DMP-1 contribute to hard tissue mineralization and the tissues affected by each are different presumably as a result of their different expression levels. In fact, in comparison with DMP-1, the functional analysis of cell signalling by DSPP remains relatively unexplored.

Serum matrix Gla protein concentrations in patients with mild and severe acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) causes an increase in proinflammatory cytokine and acute phase protein levels. Our previous studies in AP showed the role of fetuin A as a negative acute phase protein. Matrix Gla protein (MGP), beside fetuin A, is one of the main inhibitors of extraosseous calcification. In the present preliminary study we evaluated the relationship between MGP, lipase, and inflammation in AP patients. METHODS: The study included 40 patients with AP of diverse severity (28 mild, 12 severe), assessed during the early phase of AP (day 1 - day 7 of hospitalization). The concentration of MGP, fetuin A, polymorphonuclear elastase (PMN-elastase), interleukin 6 (IL-6), interleukin 18 (IL-18), hepatocyte growth factor (HGF), high sensitivity tumor necrosis factor alpha (hs TNFalpha), soluble receptor of tumor necrosis factor II (sTNFRII), and neopterin were measured by ELISA kits; albumin, lipase and amylase were measured on a Modular P Chemistry Analyser (Roche Diagnostica, Germany); procalcitonin (PCT) was measured using the LUMItest PCT (Brahms, Germany), and serum amyloid A (SAA) and high sensitivity C-reactive protein (hs CRP) were measured using an immunonephelometric method on a Nephelometer BNII (Siemens Healthcare, Germany). RESULTS: MGP positively correlated with lipase activity (R = 0.64; p < 0.05) on day 1 after admission to hospital. Lower MGP levels were consistent with higher intensity of inflammation, as MGP significantly (p < 0.05) inversely correlated with IL-6 (R = -0.48 on day 3; R = -0.46 on day 5 and R = -0.52 on day 7 after admission), IL-18 (R = - 0.55; R = -0.60; R = -0.48 on day 1, day 3, and day 5, respectively), HGF (R = -0.58 on day 3), hs TNFalpha (R = -0.45 on day 1 and R = -0.64 on day 5), its soluble receptor sTNFRII (R = -0.63; R = -0.61; R = -0.59 on day 3, day 5, and day 7, respectively), hs CRP (R = -0.76 on day 1 and R = -0.83 on day 5), PCT (R = -0.62 on day 1 and R = -0.59 on day 7), SAA (R = -0.45 on day 5) as well as with neopterin (R = -0.52 on day 1 after admission). MGP levels dropped simultaneously with fetuin A (R = 0.50 on day 3; R = 0.60 on day 5 and R = 0.63 on day 7) and albumin concentrations (R = 0.51; R = 0.70; R = 0.94 on day 1, day 5, and day 7 day after admission, respectively). There was a relationship between lipase activity and MGP concentration on day 1 of hospitalization (R = 0.64; p < 0.05). CONCLUSIONS: Our preliminary results indicate that the MGP level correlated negatively with all of the proinflammatory cytokines and acute phase proteins studied in patients with AP, and positively with lipase, fetuin A, and albumin measurements. These findings may indicate the role of MGP in calcium and phosphate metabolism disturbances in the course of AP.

[What does Reelin actually do in the developing brain?].

In mammalian brains, most neuronal cells form a layer structure. This structure is established thorough correct migration of neuronal cells, and underlies proper functions of the brain. It is thus important to understand the molecular mechanism that regulates neuronal migration and layer formation. Reelin is a large secreted protein essential for neuronal migration and layer formation in mammals. Since its identification in 1995, a number of models and hypotheses have been proposed regarding Reelin function. However, tbe primary role of Reelin in the developing brain and its underlying molecular mechanism still remain unresolved. In this review, I try to summarize what is known and what we can infer about Reelin. I also mention why studying Reelin is difficult.

Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

[Axonal growth inhibition by chondroitin sulfate proteoglycans in the central nervous system]. / Inhibición del crecimiento axonal por proteoglicanos de condroitin sulfato en el sistema nervioso central.

Chondroitin sulphate proteoglycans (CSPG) are components of the extracellular matrix, consisting of peptides chemically attached covalently to chains of glycosaminoglycans. There are 4 families of CSPG including lecticans, which are found mainly in the central nervous system (CNS) of vertebrates. In vitro studies have shown a negative effect of these proteoglycans on axonal growth, mediated by depolymerization of actin filaments in the neuronal cytoskeleton. In some neurodegenerative diseases, and especially after traumatic injuries of adult CNS, there are increased levels of CSPG expression. Axonal growth inhibition by CSPG has been observed also in vivo, and therefore a strategy aimed to counteract the inhibition of axonal growth might lead to new therapies designed to restore neural circuits. There is compelling in vivo evidence that CSPG degradation by Chondroitinase ABC allows both axonal growth and functional recovery in models of injury in the mammalian CNS. These data suggest that manipulation of the response to damage could result in effective ways to promote recovery of nerve functions in neurological disorders that affect humans, such as spinal cord lesions or Parkinson disease.

Coagulation meets calcification: the vitamin K system.

Morbidity and mortality are massively increased in patients with chronic kidney disease (CKD) and patients with end-stage renale disease (ESRD). Bone disease (renal osteodystrophy) and vascular disease (accelerated arteriosclerosis) are two typical entities contributing to this excess morbidity and mortality. Vitamin K and vitamin K-dependent-proteins play pivotal roles in the physiology of mineralization and in preventing ectopic calcification: two of these vitamin K-dependent-proteins are osteocalcin (regulating bone mineralization) and matrix-Gla protein (MGP, local calcification inhibitor in the vessel wall). Vitamin K deficiency impairs the physiological function of osteocalcin and MGP and, therefore, presumably contributes to bone demineralisation and vascular calcification (the so-called calcification paradox). In this context, the usage of vitamin K antagonists for long-term oral anticoagulation therapy might be risky especially in CKD patients exhibiting a high background level of vascular calcification. We present a summary of data describing the potential role of vitamin K deficiency and supplementation in bone and vascular disease in patients with CKD or ESRD.

Vascular calcification: lessons from scientific models.

Patients with chronic kidney disease have increased cardiovascular mortality from a combination of increased atherosclerotic disease, left ventricular hypertrophy and increased prevalence of vascular calcification (VC). Previously VC was thought to be a passive process which involved the deposition of calcium and phosphate into the vessel wall. However, recent studies have shown that VC is a highly regulated, cell-mediated process similar to bone formation, in that it is associated with expression of bone-related proteins, such as type I collagen and alkaline phosphatase. Animal and in vitro models of VC have shown that a multitude of factors including phosphate, matrix gla protein (MGP) and fetuin are involved in regulating VC. Certain factors induce calcification whereas others inhibit the process. Despite these insights, it is still not fully known how VC is regulated and a treatment for VC remains elusive. Ongoing research will hopefully elucidate these mechanisms and thereby produce targets for future therapeutic intervention. This review will highlight some of the scientific models of VC and how they have increased the understanding of this complex process.

New genes controlling human reproduction and how you find them.

The neuroendocrine control of reproduction in all mammals is governed by a hypothalamic neural network of approximately 1,500 gonadotropin releasing hormone (GnRH) secreting neurons that control activity of the reproductive axis across life. Recently, the syndrome of human GnRH deficiency, either with anosmia, termed Kallmann Syndrome (KS), or with a normal sense of smell, termed normosmic Idiopathic Hypogonadotropic Hypogonadism (nIHH), have proven important disease models that have revealed much about the abnormalities that can befall the GnRH neurons as they differentiate, migrate, form networks, mature and senesce. Mutations in several genes responsible for these highly coordinated developmental processes have thus been unearthed by the study of this prismatic disease model. This paper discusses several of the more important discoveries in this rapidly evolving field and puts them into a developmental and physiologic context.

Increased truncated TrkB receptor expression and decreased BDNF/TrkB signaling in the frontal cortex of reeler mouse model of schizophrenia.

Heterozygous reeler mouse has been used as an animal model for schizophrenia based on several neuropathological and behavioral abnormalities homologous to schizophrenia. Since some of these abnormalities are primarily associated with altered BDNF signaling we investigated BDNF signaling in the frontal cortex of reeler mice in order to shed some light on the neuropathology and treatment of schizophrenia. BDNF, TrkB receptor isoforms (full-length and truncated), reelin, GAD67, GAD65, p75NTR, and NRH-2 levels were measured in the frontal cortex samples from reeler (B6C3Fe a/a-Reln rl/+) and wild-type (WT) mice. BDNF protein levels were significantly higher in reeler compared to WT. The protein levels of full-length TrkB were not altered in reeler mice, but both mRNA and protein levels of truncated TrkB were significantly higher. Protein analysis showed that TrkB activity, as indicated by the levels of tyrosine-phosphorylated TrkB, was lower in reeler mice. We did not find any significant change in the levels of p75NTR and NRH-2, regulatory proteins of TrkB signaling, in the reeler mice. Furthermore, we found significant reduction in reelin and GAD67 expressions, but not GAD65 expression in reeler compared to WT mice. In summary, molecular processes associated with defective BDNF signaling in reeler mice provide new therapeutic targets for neuroprotective pharmacotherapy for schizophrenia.