microRNA-622 upregulates cell cycle process by targeting FOLR2 to promote CRC proliferation.

BACKGROUND: Epigenetic alterations contribute greatly to the development and progression of colorectal cancer, and effect of aberrant miR-622 expression is still controversial. This study aimed to discover miR-622 regulation in CRC proliferation. METHODS: miR-622 expression and prognosis were analyzed in clinical CRC samples from Nanfang Hospital. miR-622 regulation on cell cycle and tumor proliferation was discovered, and FOLR2 was screened as functional target of miR-622 using bioinformatics analysis, which was validated via dual luciferase assay and gain-of-function and loss-of-function experiments both in vitro and in vivo. RESULTS: miR-622 overexpression in CRC indicated unfavorable prognosis and it regulated cell cycle to promote tumor growth both in vitro and in vivo. FOLR2 is a specific, functional target of miR-622, which negatively correlates with signature genes in cell cycle process to promote CRC proliferation. CONCLUSIONS: miR-622 upregulates cell cycle process by targeting FOLR2 to promote CRC proliferation, proposing a novel mechanism and treatment target in CRC epigenetic regulation of miR-622.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content.

BACKGROUND: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. METHODS: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40). RESULTS: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). CONCLUSION: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Association of periconceptional folate supplements and FOLR1 and FOLR2 gene polymorphisms with risk of congenital heart disease in offspring: A hospital-based case-control study. / æ¯äº²å›´å­•æœŸæœç”¨å¶é ¸åŠFOLR1和FOLR2åŸºå› å¤šæ€æ€§ä¸Žå­ä»£å ˆå¤©æ€§å¿ƒè„ç— å ³è”çš„ç— ä¾‹å¯¹ç §ç ”ç©¶.

OBJECTIVES: Maternal periconceptional folic acid supplement is by far the most effective primary prevention strategy to reduce the incidence of congenital heart disease (CHD) in offspring. It was revealed that the underlying mechanisms are complex, including a combination of genetic and environmental factors. The purpose of this study is to investigate the association between periconceptional folic acid supplement, the genetic polymorphisms of maternal folic acid receptor 1 gene (FOLR1) and folic acid receptor 2 gene (FOLR2) and the impact of their interaction on the risk of CHD in offspring, and to provide epidemiological evidence for individualized folic acid dosing in hygienic counseling. METHODS: A case-control study on 569 mothers of CHD infants and 652 mothers of health controls was performed. The interesting points were periconceptional folate supplements, single nucleotide polymorphisms (SNPs) of maternal FOLR1 gene and FOLR2 gene. RESULTS: Mothers who took folate in the periconceptional period were observed a decreased risk of CHD [adjusted odds ratio (aOR)=0.58, 95% CI 0.35 to 0.95]. Our study also found that polymorphisms of maternal FOLR1 gene at rs2071010 (G/A vs G/G: aOR=0.67, 95% CI 0.47 to 0.96) and FOLR2 gene at rs514933 (T/C vs T/T: aOR=0.60, 95% CI 0.43 to 0.84; C/C vs T/T: aOR=0.55, 95% CI 0.33 to 0.90; the dominant model: T/C+ C/C vs T/T: aOR=0.59, 95% CI 0.43 to 0.81; and the addictive model: C/C vs T/C vs T/T: aOR=0.70, 95% CI 0.56 to 0.88) were significantly associated with lower risk of CHD [all P<0.05, false discovery rate P value (FDR_P)<0.1]. Besides, significant interaction between periconceptional folate supplements and rs2071010 G→A (aOR=0.59, 95% CI 0.41-0.86) and rs514933 T→C (aOR=0.52, 95% CI 0.37 to 0.74) on CHD risk were observed (all P<0.05, FDR_P<0.1). CONCLUSIONS: Periconceptional folate supplements, polymorphisms of FOLR1 gene and FOLR2 gene and their interactions are significantly associated with risk of CHD. However, more studies in different ethnic populations with a larger sample and prospective designs are required to confirm our findings.

Targeted delivery of mPGES-1 inhibitors to macrophages via the folate receptor-ß for inflammatory pain.

Activated macrophages overexpress the folate receptor ß (FR-ß) that can be used for targeted delivery of drugs conjugated to folic acid. FR-expressing macrophages contribute to arthritis progression by secreting prostaglandin E2 (PGE2). Non-steroidal anti-inflammatory drugs (NSAIDs) block PGs and thromboxane by inhibiting the cyclooxygenase (COX) enzymes and are used for chronic pain and inflammation despite their well-known toxicity. New NSAIDs target an enzyme downstream of COXs, microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of mPGES-1 in inflammatory macrophages promises to retain NSAID efficacy while limiting toxicity. We conjugated a potent mPGES-1 inhibitor, MK-7285, to folate, but the construct released the drug inefficiently. Folate conjugation to the primary alcohol of MK-7285 improved the construct's stability and the release of free drug. Surprisingly, the drug-folate conjugate potentiated PGE2 in FR-positive KB cells, and reduced PGE2 in macrophages independently of the FR. Folate conjugation of NSAIDs is not an optimal strategy for targeting of macrophages.

Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.

Autistic traits and components of the folate metabolic system: an explorative analysis in the eastern Indian ASD subjects.

Objectives: Proper metabolism of the folate is crucial for maintaining DNA integrity, chromosome structure, methylation, as well as gene expression, and thus, folate is speculated to contribute to the etiology of different disorders. Since the etiology of autism spectrum disorder (ASD) is believed to be influenced by both genetic and environmental factors, we hypothesized that functional single nucleotide polymorphisms (SNPs) affecting folate metabolic pathway may have a causal role in the etiology of ASD. Methods: We analyzed three SNPs, rs2071010, rs2298444 and rs1801198 (in the folate receptor 1, folate receptor 2 and transcobalamin 2, respectively), in 867 ethnically matched subjects including 206 ASD probands and 286 controls. Plasma vitamin B6 and folate were measured in age-matched probands and controls. Results: ASD probands showed a higher frequency of rs2298444 'A' allele (P = 0.01) and genotypes with 'A' allele (P = 0.03) when compared with the controls. rs1801198 'C' allele and 'CG' genotype also showed higher occurrence in the probands (P = 0.009 and 0.005, respectively). Gender-based stratified analysis revealed a significant higher frequency of rs2298444 'A' allele (P = 0.003), genotypes with rs2298444 'A' allele (P = 0.003) and rs1801198 CG (P = 0.001) in the male probands. Studied variants also showed statistically significant associations with ASD-associated traits measured by the Childhood Autism Rating Scale. ASD subjects exhibited gross deficiency in vitamin B6 level when compared with age-matched controls (P < 0.001), which correlated with risk genetic variants. Discussion: We infer from this pioneering study on eastern Indian subjects that vitamin B6 deficiency, along with risk gene variants, may affect ASD-associated symptoms, warranting further investigation in large cohorts.

Folate receptor-beta expression as a diagnostic target in human & rodent nonalcoholic steatohepatitis.

INTRODUCTION: Nonalcoholic steatohepatitis (NASH) afflicts 20-36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-ß) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-ß protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-ß imaging agent to the liver of a rodent NASH model using FR-ß. METHODS: Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-ß protein. The FR-ß-targeted agent, Etarfolatide was injected into MCD and MC + -fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-ß expression across the stages of human NAFLD from normal to NASH was assessed. RESULTS: FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8 weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-ß in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-ß protein when compared to normal liver. FR-ß transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples. CONCLUSION: The findings in this study indicate that FR-ß expression in NASH may be harnessed to target agents directly to the liver.

Integrative analysis of oncogenic fusion genes and their functional impact in colorectal cancer.

BACKGROUND: Fusion genes are good candidates of molecular targets for cancer therapy. However, there is insufficient research on the clinical implications and functional characteristics of fusion genes in colorectal cancer (CRC). METHODS: In this study, we analysed RNA sequencing data of CRC patients (147 tumour and 47 matched normal tissues) to identify oncogenic fusion genes and evaluated their role in CRC. RESULTS: We validated 24 fusion genes, including novel fusions, by three algorithms and Sanger sequencing. Fusions from most patients were mutually exclusive CRC oncogenes and included tumour suppressor gene mutations. Eleven fusion genes from 13 patients (8.8%) were determined as oncogenic fusion genes by analysing their gene expression and function. To investigate their oncogenic impact, we performed proliferation and migration assays of CRC cell lines expressing fusion genes of GTF3A-CDK8, NAGLU- IKZF3, RNF121- FOLR2, and STRN-ALK. Overexpression of these fusion genes increased cell proliferation except GTF3A-CDK8. In addition, overexpression of NAGLU-IKZF3 enhanced migration of CRC cells. We demonstrated that NAGLU-IKZF3, RNF121-FOLR2, and STRN-ALK had tumourigenic effects in CRC. CONCLUSION: In summary, we identified and characterised oncogenic fusion genes and their function in CRC, and implicated NAGLU-IKZF3 and RNF121-FOLR2 as novel molecular targets for personalised medicine development.

Genetic assessment and folate receptor autoantibodies in infantile-onset cerebral folate deficiency (CFD) syndrome.

INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3´phosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and ß (hFRß).

Human folate receptors (hFRα and hFRß) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requires a large amount of hFRs. However, it is difficult to prepare active forms of hFRs from prokaryotic cells because of their high content of cysteine residues that form disulfide bonds. Here, we prepared active forms of hFRα and hFRß from inclusion bodies of Escherichia coli. The crucial steps in our preparation were intensive washing of the inclusion bodies to remove impurities derived from E. coli and gradual dropping of solubilized hFRs into refolding buffers to correctly reform disulfide bonds. The binding activity of prepared hFRs to folate was confirmed by biolayer interferometry measurements. Finally, we successfully prepared the active form of 2.52 mg hFRα and 2.4 mg hFRß from 10 g of E. coli cell bodies.

Silencing the FOLR2 Gene Inhibits Cell Proliferation and Increases Apoptosis in the NCI-H1650 Non-Small Cell Lung Cancer Cell Line via Inhibition of AKT/Mammalian Target of Rapamycin (mTOR)/Ribosomal Protein S6 Kinase 1 (S6K1) Signaling.

BACKGROUND The FOLR2 gene encodes folate receptor-beta (FR-beta), which is expressed by tumor-associated macrophages. The effects of FOLR2 gene expression in non-small cell lung cancer (NSCLC) remains unknown. This study aimed to investigate the effects of FOLR2 gene expression and gene silencing in human NSCLC cell lines and normal human bronchial epithelial (HBE) cells in vitro. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the expression of the FOLR2 gene, cell cycle and apoptosis-associated genes in normal HBE cells and the NSCLC cell lines, A549, NCI-H1299, NCI-H1650, and NCI-H460. Using small interfering RNA (siRNA), or silencing RNA, FOLR2 gene silencing was performed for NCI-H1650 cells. Cell counting kit-8 (CCK-8) was used to measure cell viability. Cell cycle and apoptosis were determined using flow cytometry. Western blot evaluated the expression of Akt, mTOR, and S6K1 signaling. RESULTS Expression of the FOLR2 gene was increased in NSCLC cells compared with normal HBE cells. Silencing of the expression of the FOLR2 gene in NCI-H1650 cells reduced cell viability, increased cell apoptosis, and arrested cells in the G1 phase of the cell cycle, decreased the expression of cyclin D1, upregulated expression of cell cycle inhibitors, p21 and p27, upregulated the expression of Bax/Bcl-2, and inhibited phosphorylation of AKT, mTOR, and S6K1. CONCLUSIONS Silencing of the FOLR2 gene inhibited phosphorylation of AKT, mTOR, and S6K1, inhibited cell proliferation and increased apoptosis in the NCI-H1650 human NSCLC cell line.

Targeting of folate receptor ß on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor ß (FRß) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRß (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRß as well as endogenous FRß(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRß(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRß expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRß expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRß is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA.

Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells.

Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency.Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo.Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1.

Mutations in folate transporter genes and risk for human myelomeningocele.

The molecular mechanisms linking folate deficiency and neural tube defect (NTD) risk in offspring remain unclear. Folate transporters (SLC19A1, SLC46A1, SLC25A32, and FOLH1) and folate receptors (FOLR1, FOLR2, and FOLR3) are suggested to play essential roles in transporting folate from maternal intestinal lumen to the developing embryo. Loss of function variants in these genes may affect folate availability and contribute to NTD risk. This study examines whether variants within the folate transporter and receptor genes are associated with an increased risk for myelomeningocele (MM). Exons and their flanking intron sequences of 348 MM subjects were sequenced using the Sanger sequencing method and/or next generation sequencing to identify variants. Frequencies of alleles of single nucleotide polymorphisms (SNPs) in MM subjects were compared to those from ethnically matched reference populations to evaluate alleles' associated risk for MM. We identified eight novel variants in SLC19A1 and twelve novel variants in FOLR1, FOLR2, and FOLR3. Pathogenic variants include c.1265delG in SLC19A1 resulting in an early stop codon, four large insertion deletion variants in FOLR3, and a stop_gain variant in FOLR3. No new variants were identified in SLC46A1, SLC25A32, or FOLH1. In SLC19A1, c.80A>G (rs1051266) was not associated with our MM cohort; we did observe a variant allele G frequency of 61.7%, higher than previously reported in other NTD populations. In conclusion, we discovered novel loss of function variants in genes involved in folate transport in MM subjects. Our results support the growing evidence of associations between genes involved in folate transport and susceptibility to NTDs.

Folate-targeted nanoparticles for rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and showed specifically express a receptor for the vitamin folic acid (FA), folate receptor ß (FRß). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions. FROM THE CLINICAL EDITOR: Rheumatoid arthritis is a debilitating autoimmune disease of the joints which affects many people worldwide. Up till now, there is a lack of optimal therapy against this disease. In this review article, the authors outlined in depth the current mechanism of disease for rheumatoid arthritis and described the latest research in using folic acid-targeted nanoparticles to target synovial macrophages in the fight against rheumatoid arthritis.

Molecular imaging of folate receptor ß-positive macrophages during acute lung inflammation.

Characterization of markers that identify activated macrophages could advance understanding of inflammatory lung diseases and facilitate development of novel methodologies for monitoring disease activity. We investigated whether folate receptor ß (FRß) expression could be used to identify and quantify activated macrophages in the lungs during acute inflammation induced by Escherichia coli LPS. We found that FRß expression was markedly increased in lung macrophages at 48 hours after intratracheal LPS. In vivo molecular imaging with a fluorescent probe (cyanine 5 polyethylene glycol folate) showed that the fluorescence signal over the chest peaked at 48 hours after intratracheal LPS and was markedly attenuated after depletion of macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5-conjugated folate as FRß(+) interstitial macrophages and pulmonary monocytes, which coexpressed markers associated with an M1 proinflammatory macrophage phenotype. These findings were confirmed using a second model of acute lung inflammation generated by inducible transgenic expression of an NF-κB activator in airway epithelium. Using CC chemokine receptor 2-deficient mice, we found that FRß(+) macrophage/monocyte recruitment was dependent on the monocyte chemotactic protein-1/CC chemokine receptor 2 pathway. Together, our results demonstrate that folate-based molecular imaging can be used as a noninvasive approach to detect classically activated monocytes/macrophages recruited to the lungs during acute inflammation.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

Folate receptor (FR)-ß has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.

Purifying selection against gene conversions in the folate receptor genes of primates.

We characterized the gene conversions between the human folate receptor (FOLR) genes and those of five other primate species. We found 26 gene conversions having an average length of 534 nucleotides. The length of these conversions is correlated with sequence similarity, converted regions have a higher GC-content and the average size of converted regions from a functional donor to another functional donor is significantly smaller than the average size from a functional donor to a pseudogene. Furthermore, the few conversions observed in the FOLR1 and FOLR2 genes did not change any amino acids in their coding regions and did not affect their promoter regions. In contrast, the promoter and coding regions of the FOLR3 gene are frequently converted and these conversions changed many amino acids in marmoset. These results suggest that purifying selection is limiting the functional impact that frequent gene conversions have on functional folate receptor genes.

Imaging atherosclerotic plaque inflammation via folate receptor targeting using a novel 18F-folate radiotracer.

Folate receptor ß (FR-ß) is overexpressed on activated, but not resting, macrophages involved in a variety of inflammatory and autoimmune diseases. A pivotal step in atherogenesis is the subendothelial accumulation of macrophages. In nascent lesions, they coordinate the scavenging of lipids and cellular debris to define the likelihood of plaque inflammation and eventually rupture. In this study, we determined the presence of FR-ß-expressing macrophages in atherosclerotic lesions by the use of a fluorine-18-labeled folate-based radiotracer. Human endarterectomized specimens were used to measure gene expression levels of FR-ß and CD68. Increased FR-ß and CD68 levels were found in atherosclerotic plaques compared to normal artery walls by quantitative real-time polymerase chain reaction. Western blotting and immunohistochemistry demonstrated prominent FR-ß protein levels in plaques. FR-ß-positive cells colocalized with activated macrophages (CD68) in plaque tissue. Carotid sections incubated with 3'-aza-2'-[18F]fluorofolic acid displayed increased accumulation in atherosclerotic plaques through in vitro autoradiography. Specific binding of the radiotracer correlated with FR-ß-expressing macrophages. These results demonstrate high FR-ß expression in atherosclerotic lesions of human carotid tissue correlating with CD68-positive macrophages. Areas of high 3'-aza-2'-[18F]fluorofolic acid binding within the lesions represented FR-ß-expressing macrophages. Selectively targeting FR-ß-positive macrophages through folate-based radiopharmaceuticals may be useful for noninvasive imaging of plaque inflammation.

Methotrexate normalizes up-regulated folate pathway genes in rheumatoid arthritis.

OBJECTIVE: The folate antagonist methotrexate (MTX) is an anchor drug in the treatment of rheumatoid arthritis (RA), but its mechanism of action with regard to the impact on folate metabolism remains elusive. The aim of the present study was to investigate the cellular pharmacologic impact of MTX on peripheral blood cells, by comparing MTX-treated RA patients to MTX-naive RA patients and healthy controls. METHODS: Gene expression microarray data were used to investigate the expression of 17 folate pathway genes by peripheral blood cells from a cohort of 25 RA patients treated with MTX, 10 MTX-naive RA patients starting treatment with MTX, and 15 healthy controls (test cohort). Multiplex real-time polymerase chain reaction was used to validate the results in an independent cohort, consisting of 151 RA patients treated with MTX, 28 MTX-naive RA patients starting treatment with MTX, and 24 healthy controls (validation cohort). RESULTS: Multiple folate metabolism-related genes were consistently and significantly altered between the 3 groups in both cohorts. Concurrent with evidence of an immune-activation gene signature in MTX-naive RA patients, significant up-regulation of the folate-metabolizing enzymes γ-glutamyl hydrolase and dihydrofolate reductase, as well as the MTX/folate efflux transporters ABCC2 and ABCC5, was observed in the MTX-naive RA group compared to healthy controls. Strikingly, MTX treatment of RA patients normalized these differential gene expression levels to the levels observed in healthy controls. CONCLUSION: These results suggest that under inflammatory conditions, basal folate metabolism in the peripheral blood cells of RA patients is markedly up-regulated, and treatment with MTX restores folate metabolism to normal levels. Identification of this novel gene signature provides insight into the mechanism of action of MTX, thus paving the way for development of novel folate metabolism-targeted therapies.