Multitarget, multiagent PLGA nanoparticles for simultaneous tumor eradication and TME remodeling in a melanoma mouse model.

Despite the fact that chemoimmunotherapy has emerged as a key component in the era of cancer immunotherapy, it is challenged by the complex tumor microenvironment (TME) that is jam-packed with cellular and non-cellular immunosuppressive components. The aim of this study was to design a nanoparticulate system capable of sufficiently accumulating in the tumor and spleen to mediate local and systemic immune responses, respectively. The study also aimed to remodel the immunosuppressive TME. For such reasons, multi-functional polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) were engineered to simultaneously eradicate the cancer cells, silence the tumor-associated fibroblasts (TAFs), and re-educate the tumor-associated macrophages (TAMs) using doxorubicin, losartan, and metformin, respectively. These agents were also selected for their ability to tip the balance of the splenic immune cells towards immunostimulatory phenotypes. To establish TAM and TAF cultures, normal macrophages and fibroblasts were incubated with B16F10 melanoma cell (Mel)-derived secretome. Drug-loaded PLGA NPs were prepared, characterized, and tested in the target cell types. Organ distribution of fluorescein-loaded PLGA NPs was evaluated in a mouse model of melanoma. Finally, the local and systemic effects of different combination therapy programs were portrayed. The in vitro studies showed that the drug-loaded PLGA NPs could significantly ablate the immunosuppressive nature of Mel and skew TAMs and TAFs towards more favorable phenotypes. While in vivo, PLGA NPs were proven to exhibit long blood circulation time and to localize preferentially in the tumor and the spleen. The combination of either metformin or losartan with doxorubicin was superior to the monotherapy, both locally and systemically. However, the three-agent combo produced detrimental effects in the form of compromised well-being, immune depletion, and metastasis. These findings indicate the potential of TME remodeling as means to prime the tumors for successful chemoimmunotherapy. In addition, they shed light on the importance of the careful use of combination therapies and the necessity of employing dose-reduction strategies. D-NPs doxorubicin-loaded NPs, M-NPs metformin-loaded NPs, L-NPs losartan-loaded NPs, TAMs tumor-associated macrophages, TAFs tumor-associated fibroblasts, PD-L1 programmed death ligand 1, TNF-α tumor necrosis factor alpha, TGF-ß transforming growth factor beta, CD206/40/86 cluster of differentiation 206/40/86, α-SMA alpha-smooth muscle actin, MMPs matrix metalloproteases.

An intronic DHCR7 genetic polymorphism associates with vitamin D serum level and incidence of acute coronary syndrome.

INTRODUCTION: Vitamin D deficiency has been linked to cardiovascular pathologies including acute coronary syndrome (ACS). Polymorphisms in vitamin D associated genes have been confounding to vitamin D serum levels and pathological predispositions. 7-hydrocholesterol is a common precursor in cholesterol and vitamin D synthesis. DHCR7/NADSYN1 genetic locus expresses 7-hydrocholesterol reductase (DHCR7), an enzyme that recruits 7-hydrocholesterol in cholesterol biosynthesis, and NAD synthetase 1 (NADSYN1), which participates in the hydroxylation of 25 hydroxyvitamin D. AIM: This study aims to correlate two polymorphisms in the DHCR7/NADSYN1 genetic locus with levels of circulatory vitamin D and the presentation of ACS in an Egyptian population. METHODS: In a case control study, 189 ACS patients and 106 healthy control subjects were genotyped for SNPs rs11606033 of the DHCR7 gene and rs2276360 of the NADSYN1 gene using the amplification-refractory mutation system (ARMS). The levels of 25(OH)D2 and 25(OH)D3 were measured using an in-house developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based protocol. RESULTS: ACS patients have significantly lower levels of circulating vitamin D in comparison to healthy controls. Allele A of the DHCR7 polymorphism was found to correlate with serum vitamin D deficiency and incidence of ACS classes: NSTEMI, STEMI and unstable angina, when compared to allele G. On the other hand, the NADSYN1 polymorphism rs2276360 correlated with serum 25(OH)D3 deficiency. Yet, no significant correlation was found with incidences of ACS. CONCLUSION: We conclude that rs11606033, which is an intronic SNP between exon 4 and exon 5 of the DHCR7 gene, influences vitamin D serum abundance and more importantly ACS incidence.

The association of megalin and cubilin genetic variants with serum levels of 25-hydroxvitamin D and the incidence of acute coronary syndrome in Egyptians: A case control study.

Megalin and cubilin are two receptors that mediate endocytosis of 25-hydroxyvitamin D (25(OH)D) for its final activation by hydroxylation. The aim of the present study was to evaluate the association of polymorphisms in megalin (rs2075252 and rs4668123) and cubilin (rs1801222 and rs12766939) with the circulating serum levels of 25(OH)D and with the early incidence of acute coronary syndrome (ACS) in Egyptians. The study included 328 subjects; 185 ACS patients aged between 27 and 60 years, and 143 healthy age-matched controls. Genotyping of cubilin rs12766939 Single Nucleotide Polymorphism (SNP) was performed using Real-Time Polymerase Chain Reaction (qPCR) and for megalin rs4668123 and rs2075252 and cubilin rs1801222 by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP). 25(OH)D levels were measured by Ultra Performance Liquid Chromatography- Tandem Mass Spectroscopy (UPLC-MS/MS). Results showed that vitamin D deficiency was highly linked to ACS incidence (P < 0.0001). The megalin rs4668123 CC, cubilin rs1801222 GG and cubilin rs12766939 GG + GA genotypes are associated with a higher ACS incidence and can be considered risk factors, according to Chi-squared test (P = 0.0003, 0.0442, 0.013 respectively). Conversely, the megalin rs2075252 SNP was not associated with increased ACS incidence. However, after performing multiple logistic regression analysis, only the megalin rs4668123 SNP was considered an independent ACS risk factor. Furthermore, the megalin rs4668123 CC genotype was associated with lower 25(OH)D levels (P = 0.0018). In conclusion, megalin rs4668123 (CC) was linked to lower 25(OH)D levels and can be considered an independent risk factor for incidence of ACS.

Contribution of CYP27B1 and CYP24A1 genetic variations to the incidence of acute coronary syndrome and to vitamin D serum level.

Cardiovascular diseases remain a major public health burden worldwide. It was reported that vitamin D protects the cardiovascular system through several mechanisms mainly by hindering atherosclerosis development. Genetic variations in vitamin D metabolic pathway were found to affect vitamin D levels. This study aimed at investigating the association between single nucleotide polymorphisms in genes involved in vitamin D metabolism, CYP27B and CYP24A1; 25-hydroxyvitamin D (25(OH)D) levels; and susceptibility to acute coronary syndrome (ACS). One hundred and eighty-five patients and 138 healthy controls were recruited. CYP24A1 rs2762939 was genotyped using fast real-time PCR, while CYP24A1 rs4809960 and CYP27B1 rs703842 were genotyped using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). 25(OH)D3 and 25(OH)D2 levels were measured using ultra-performance liquid chromatography tandem mass spectrum. Vitamin D level was significantly lower in patients than controls (p < 0.05). The GG genotype of rs2762939 was significantly associated with the risk of ACS development, but not correlated to the vitamin D level. rs4809960 and rs703842 genetic variations were not associated with ACS nor with 25(OH)D level. The genetic variant rs2762939 of CYP24A1 is remarkably associated with ACS. Meanwhile, the variants rs4809960 and rs703842 are not associated with ACS incidence.

AGXT2 and DDAH-1 genetic variants are highly correlated with serum ADMA and SDMA levels and with incidence of coronary artery disease in Egyptians.

Dimethylarginine aminodehydrolase (DDAH1) and alanine glyoxylate aminotransferase2 (AGXT2) are two enzymes that contribute in asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) metabolism. Hence they affect production and bioavailability of eNOS-derived nitric oxide (NO) and consequently healthy blood vessels. The major aims of the current study were to investigate the association of genetic variants of AGXT2 rs37369, AGXT2 rs16899974 and DDAH1 rs997251 SNPs with incidence of coronary artery disease (CAD) in Egyptians and to correlate these variants with the serum levels of ADMA and SDMA. The study included 150 subjects; 100 CAD patients and 50 healthy controls. Genotyping was performed by qPCR while the ADMA and SDMA concentrations were assayed by ELISA. Both serum ADMA and SDMA concentrations were significantly higher in CAD patients compared to controls (both p < 0.0001). Genotype distributions for all studied SNPs were significantly different between CAD patients and controls. Carriers of AGXT2 rs37369-T allele (CT + TT genotypes) and AGXT2 rs16899974-A allele (CA + AA genotypes) had 2.4- and 2.08-fold higher risk of having CAD than CC genotype in both SNPs (p = 0.0050 and 0.0192, respectively). DDAH1 rs997251 TC + CC genotypes were associated with 2.3-fold higher risk of CAD than TT genotype (p = 0.0063). Moreover, the AGXT2 rs37369 TT and AGXT2 rs16899974 AA genotypes were associated with the highest serum ADMA and SDMA while DDAH1 rs997251 CC genotype was associated with the highest ADMA. AGXT2 rs37369-T, AGXT2 rs16899974-A, and DDAH1 rs997251-C alleles represent independent risk factors for CAD in the Egyptians.

Genetic Variants of CYP2R1 Are Key Regulators of Serum Vitamin D Levels and Incidence of Myocardial Infarction in Middle-Aged Egyptians.

BACKGROUND: Myocardial Infarction (MI) is one of the leading causes of morbidity and mortality in Egypt and worldwide. Vitamin D deficiency has long been linked to incidence of cardiovascular diseases. Several factors were reported to contribute to serum vitamin D level including exposure to sunlight. However, genetic variations in the vitamin D metabolic pathways have also been considered as strong determinants of vitamin D levels. CYP2R1 is the major 25-hydroxylase enzyme that is responsible for the 1st activation step of vitamin D. OBJECTIVE: to investigate the contribution of polymorphisms in CYP2R1 gene to vitamin D deficiency and incidence of MI in Egyptians. METHODS: The study included 323 subjects; 185 MI patients and 138 healthy controls. Serum 25OHD3, 25OHD2 and total 25OHD levels were measured using LC-MS/MS. SNPs rs2060793 and rs1993116 were determined by polymerase chain reaction - restriction fragment length polymorphism (PCRRFLP) which is considered one of the most commonly used techniques in genotyping. SNP rs10766197 was detected using TaqMan allele discrimination assay. RESULTS: Serum 25OHD3, 25OHD2 and total 25OHD levels were found to be significantly lower in MI patients than controls. The three studied SNPs were associated with significantly different total 25OHD levels and their genotype distributions differed significantly between MI patients and controls where the high risk genotypes were AG/AA for rs2060793, AG/GG for rs1993116 and AG/AA for rs10766197. Additionally, the concurrent presence of high risk genotypes of the three studied SNPs rendered those individuals at extremely higher risk for MI than each individual SNP (OR 14.1, 95% CI (3.1-64.7), p-value = < 0.0001). CONCLUSIONS: Genetic variants of CYP2R1 are key determinants of serum 25OHD levels and are highly associated with MI risk.

Independent assortment of GC gene polymorphism (rs2282679) and 25-hydroxyvitamin D levels in coronary artery disease.

Coronary artery disease (CAD) remains a major public health burden. Emerging research has suggested an association between vitamin D insufficiency and CAD. Vitamin D binding protein (VDBP) is the primary vitamin D carrier and many of its genetic polymorphisms are able to induce the expression of proteins with different affinities for the vitamin, which in turn might affect its serum levels and CAD incidence. One hundred and twelve male patients, aged between 35 and 50 years, with verified CAD and 109 age- and sex-matched controls were recruited. Genotyping was performed by the TaqMan allelic discrimination assay and plasma 25(OH)D levels were assessed by HPLC-UV. Serum parathyroid hormone (s-PTH) and VDBP levels were measured using ELISA. s-25(OH)D levels in CAD patients were significantly lower than in the controls, whereas s-PTH levels were significantly higher in the CAD patients than in the controls. There was no significant difference in the distribution of GC genotypes among both groups. s-25(OH)D showed a weak inverse correlation with s-PTH levels. Serum levels of vitamin D and PTH are highly correlated with CAD incidence. However, the s-VDBP level is associated neither with disease outcome nor with vitamin D status. The GC gene variant has no effect on 25(OH)D levels.

T-786C variation in the promoter sequence of human eNOS gene markedly influences its expression level.

This study investigated the role of the T-786C polymorphism (SNP) in the 5'-flanking sequence of the endothelial nitric oxide synthase gene (eNOS) on its expression level in vascular endothelium with the ultimate goal of shedding more light on the mechanisms by which genetic variations of eNOS might affect the vascular level of nitric oxide (NO). Sequences in the 5'-flanking region of eNOS gene were PCR-amplified using genomic DNA templates isolated from blood samples collected from cardiovascular disease (CVD) patients. Two sequence-versions carrying the same SNP site were used; a short (345 bp) and an extended one (1,594 bp), numbered relative to the translational start site. All sequences were cloned into a promoter-less vector (pGL3-basic), which carries the firefly luciferase gene as a reporter. Genotyping of the T-786C polymorphism was performed using Sanger sequencing of the insert region. Luminescence levels were then recorded 24-48 h after transfecting human endothelial cell line (EA.hy926). Three genotypes were identified in the subject samples; TT, TC, or CC. The highest expression levels associated with the TT genotype, followed by the TC genotype, then the CC genotype. The extended sequence version produced higher expression levels compared to the shorter version. Our results provide evidence that the T allele at the T-786C SNP site of the eNOS gene results in increased expression of the enzyme, and consequently might provide a protective mechanism from CVD. The extended promoter sequence of eNOS resulted in higher expression of the gene, suggesting the presence of some essential binding sites for transcription enhancing proteins.

"Desert" gene (Chr9p21) variants as novel markers for coronary artery disease.

OBJECTIVE: Previous reports have denoted to the possible link of Chr9p21 locus to the incidence of coronary artery disease (CAD). The entire core of chr9p21 is covered by "ANRIL" (Antisense noncoding RNA in INK4 Locus) and lies in a region that is free from any coding proteins; therefore, it is called the desert gene. The major objectives of this study were to examine the association of rs10757278 and rs2383206 SNPs on Chr9p21 with the incidence of CAD in the presence and absence of type 2 diabetes (T2D) in Egyptians and to correlate these genetic variants with several disease biomarkers (TC, CRP, and HbA1c). METHODS: The study subjects consisted of 150 subjects; 50 healthy controls and 100 patients that were divided into two groups; CAD patients and CAD T2D patients. The genotyping of SNPs was performed using qPCR. RESULTS: Genotype distribution for both SNPs were found to be significantly different (p=0.0009 for rs10757278 and p=0.001 for rs2383206) between patients and controls. The allele frequency was also different for rs10757278. CONCLUSION: The current study showed that rs10757278/rs2383206-G allele increases the risk for CAD in Egyptians. Moreover, AA variant appeared as a protective genotype. However, SNPs did not noticeably contribute in the elevation of TC, hs-CRP, and HbA1c in non-diabetic and diabetic CAD patients.

Investigation of brain-derived neurotrophic factor (BDNF) gene expression in hypothalamus of obese rats: Modulation by omega-3 fatty acids.

PURPOSE: The aim of this study was investigating the effect of omega-3 fatty acids (ω-3 FAs) on brain-derived neurotrophic factor (BDNF) gene expression, using in vivo and in vitro models, to unravel the potential mechanisms of polyunsaturated fatty acids use in obesity. MATERIALS AND METHODS: Twenty-nine Sprague-Dawley rats were divided into three groups; lean controls fed normal chow diet for 14 weeks, obese controls fed 60% of their diet as saturated fats for 14 weeks, and ω-3 FAs-treated rats fed 60% saturated fat diet for 14 weeks with concomitant oral administration of 400 mg/kg/day ω-3 FAs, mainly docosahexaenoic acid and EPA, from week 12 to week 14. For the in vitro experiment, hypothalamic cells from six obese rats were cultured in the presence of different concentrations of ω-3 FAs to determine its direct effect on BDNF expression. RESULTS: In vivo results showed that obesity has negative effect on BDNF gene expression in rat hypothalamus that was reversed by administration of ω-3 FAs. Obese rats showed hypercholesterolemia, hypertriglyceridemia, normoinsulinemia, hyperglycemia and hyperleptinemia. Treatment with ω-3 FAs showed significant decrease in serum total cholesterol and TAG. Also serum glucose level and HOMA index were decreased significantly. In vitro results demonstrated the increase in BDNF expression by ω-3 FAs in a dose-dependent manner. CONCLUSIONS: Obesity causes down-regulation of BDNF gene expression that can be reversed by ω-3 FAs treatment, making them an interesting treatment approach for obesity and metabolic disease.

Genetic variation in vitamin D receptor gene (Fok1:rs2228570) is associated with risk of coronary artery disease.

OBJECTIVE: The Fok1 polymorphism (rs2228570) in vitamin D receptor gene appears to be the only polymorphism influencing size of translated protein. Investigations into its association with coronary artery disease (CAD) are sparse. METHODS: Male patients (n = 98) with verified CAD were recruited alongside age- and sex-matched controls (n = 55). Genotyping was performed by PCR-RFLP and plasma 25-Hydroxyvitamin D levels were assessed by HPLC-UV. RESULTS: The C-variant (mutant) was predominantly expressed in patients compared to controls (68.9% versus 55.5%; p = 0.025). The observed genotypes were not associated with 25-Hydroxyvitamin D levels. CONCLUSION: This study presents Fok1 polymorphism as a potential genetic marker for CAD.

Vitamin D receptor gene polymorphisms (TaqI and ApaI) in relation to 25-hydroxyvitamin D levels and coronary artery disease incidence.

CONTEXT/OBJECTIVE: Previous studies have illustrated the association of the ApaI and TaqI polymorphisms of the vitamin D receptor gene, located in non-coding and coding regions, respectively, with diseases such as cancer and cardiovascular disease; however, investigating such association in Egyptian patients with coronary artery disease (CAD) has never been formerly attempted. MATERIALS AND METHODS: Male patients (n = 137), 35-50 years of age, with verified CAD, were recruited alongside age- and sex-matched controls (n = 58). Genotyping and 25-hydroxyvitamin D [25(OH)D] measurement were performed by polymerase chain reaction RFLP and HPLC, respectively. RESULTS: Comparison of the genotypic distribution of both the TaqI and ApaI polymorphisms between patients and controls yielded insignificant results (p = 0.55 and 0.7, respectively). Comparison of the allelic distribution of both polymorphisms also yielded insignificant results. The TaqI polymorphism was not found to predict 25(OH)D levels, whereas the wild-type genotype of the ApaI polymorphism was associated with greater levels of 25(OH)D (p = 0.02), taking all subjects into consideration. DISCUSSION/CONCLUSION: This study presents the ApaI and TaqI polymorphisms as non-influencing players in the pathogenesis of CAD in Egyptian males and the ability of only the ApaI polymorphism to predict 25(OH)D levels, thus warranting further investigations of the triangular relationship between the polymorphisms, 25(OH)D and CAD incidence.

Triangular relationship between single nucleotide polymorphisms in the CYP2R1 gene (rs10741657 and rs12794714), 25-hydroxyvitamin d levels, and coronary artery disease incidence.

OBJECTIVE: To investigate the relationship between the rs10741657 and rs12794714 polymorphisms in the CYP2R1 gene, 25(OH)D levels, and coronary artery disease (CAD) incidence. METHODS: In total, 134 male patients with verified CAD were recruited, alongside 109 age- and sex-matched controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, using the corresponding restriction enzyme for each polymorphism, whereas 25(OH)D levels were analyzed by HPLC-UV. RESULTS: 25(OH)D levels were significantly lower in patients. The genotypic and allelic distributions of the rs10741657 polymorphism were significantly different between patients and controls, whereas insignificant results were obtained for the rs12794714 polymorphism. Furthermore, rs10741657, but not rs12794714, predicted 25(OH)D levels. CONCLUSION: The rs10741657 polymorphism is a novel genetic marker for CAD.

Insights on vitamin D's role in cardiovascular disease: investigating the association of 25-hydroxyvitamin D with the dimethylated arginines.

Accumulating evidence has stipulated a strong correlation between vitamin D (vitD) deficiency and cardiovascular disease (CVD); however, a mechanistic link is missing. This study investigated the association of vitD with endothelial dysfunction parameters. Subjects comprised male patients with verified coronary artery disease (CAD) (n=69) and age- and sex-matched controls (n=20). 25-Hydroxyvitamin D [25(OH)D] was determined using high performance liquid chromatography with ultraviolet detection whereas asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) were determined by liquid chromatography-mass spectrometry. Nitric oxide (NO) was determined spectrophotometrically and high-sensitivity C-reactive protein (hs-CRP) was determined using enzyme-linked immunosorbent assay (ELISA). Comparison of mean 25(OH)D concentrations of patients and controls yielded a significant result (p=0.0002). 25(OH)D2 was dominant in patients whereas 25(OH)D3 was dominant in controls (p=0.003 and 0.001, respectively). Comparison of mean ADMA and SDMA concentrations of patients exhibiting normal and suboptimal vitD yielded insignificant results (p=0.692 and 0.998, respectively). Significant results were obtained from the comparison of mean hs-CRP and NO concentrations of patients exhibiting normal and suboptimal vitD (p=0.035 and 0.031, respectively). Results suggest involvement of vitD with the NO system, however not via modulation of the dimethylated arginines. A potential anti-inflammatory activity for vitD is also raised.

Effect of polymorphisms in the NADSYN1/DHCR7 locus (rs12785878 and rs1790349) on plasma 25-hydroxyvitamin D levels and coronary artery disease incidence.

BACKGROUND/AIMS: Recent genome-wide association studies have identified the rs1790349 and rs12785878 single-nucleotide polymorphisms (SNPs), present in the NADSYN1/DHCR7 locus, as an influential player on circulating 25-hydroxyvitamin D [25(OH)D] levels, which itself has been linked to various diseases including cardiovascular disease (CVD). This study investigated the association of these SNPs with CVD and 25(OH)D levels. METHODS: Sixty- three male patients with verified coronary artery disease (CAD) were recruited, as well as 31 age- and sex-matched controls. Genotyping was performed by sequencing, whereas plasma 25(OH)D levels were assessed by HPLC-UV. RESULTS: Statistical insignificance was observed in comparing the genotype distribution of patients and controls for both the rs12785878 (NADSYN1) polymorphism (p = 0.097) and the rs1790349 (DHCR7; p = 0.9). Comparison of allelic distributions of rs1790349 and rs12785878 yielded insignificant results (p = 0.7, OR: 0.58-2.6 and p = 0.14, OR: 0.88-2.85, respectively). Taking together patients and controls, both SNPs were found to influence total 25(OH)D levels (p = 0.001 and p < 0.0001) as well as 25(OH)D3 levels only in controls. CONCLUSION: This study further supports the evidence of the ability of the investigated SNPs to predict circulating 25(OH)D levels, nonetheless opposing their use as genetic markers for CAD.

Assessment of serum levels of asymmetric dimethylarginine, symmetric dimethylarginine and L-arginine in coronary artery disease.

Serum asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-arginine, and C-reactive protein (hsCRP) levels were assessed in 100 Egyptian male 35-50-year-old patients with coronary artery disease (CAD), classified into: patients under conservative medical treatment, patients directed for percutaneous coronary interventions, patients directed for coronary artery bypass graft operation and patients suffering from acute myocardial infarction. Age- and sex-matched controls (n=100) were included. Correlation between serum levels of biomarkers and dimethylarginine dimethylaminohydrolase-2 (DDAH-2) genotypes was studied. No association between biomarkers and carriage of the specific DDAH2 SNP2 (-449C/G, rs805305) genotype was detected. Further studies are required to confirm the contribution of the biomarkers in the predisposition of CAD.