High Incidence of ACE/PAI-1 in Association to a Spectrum of Other Polymorphic Cardiovascular Genes Involving PBMCs Proinflammatory Cytokines in Hypertensive Hypercholesterolemic Patients: Reversibility with a Combination of ACE Inhibitor and Statin

Abstract

Cardiovascular diseases (CVDs) are significantly high in the Lebanese population with the two most predominant forms being atherosclerosis and venous thrombosis. The purpose of our study was to assess the association of a spectrum of CVD related genes and combined state of hypertension hypercholesterolemia (HH) in unrelated Lebanese. Twelve polymorphisms were studied by multiplex PCR and reverse hybridization of DNA from 171 healthy individuals and 144 HH subjects. Two genes were significantly associated with HH: ACE (OR: 9.20, P<0.0001) and PAI-1 (OR: 2.29, P = 0.007), respectively with the occurrence of the risky alleles "Del" and "4G". The frequencies of the Del and 4G alleles were found to be 0.98 and 0.90 in the HH group versus 0.84 and 0.79 in the healthy group, respectively. Serum ACE activity and PAI-I increased significantly with Del/Del and 4G/5G genotypes. The co-expression of Del/4G(+/+) was detected in 113 out of 171 (66.0%) controls and 125 out of 144 (86.8%) HH subjects. Del/4G(-/-) was detected in only 6 (3.5%) controls and undetected in the HH group. Three venous thrombosis related genes [FV(Leiden), MTHFR(A1298C) and FXIII(V34L)] were significantly related to the prominence of the co-expression of Del/4G(+/+). A range of 2 to 8 combined polymorphisms co-expressed per subject where 5 mutations were the most detected. In Del/4G(+/+) subjects, peripheral blood mononuclear cells (PBMCs) produced significant elevated levels of IFN-γ and TNF-α contrary to IL-10, and no variations occurred for IL-4. ACE inhibitor (ramipril) in combination with statin (atorvastatin) and not alone reversed significantly the situation. This first report from Lebanon sheds light on an additional genetic predisposition of a complex spectrum of genes involved in CVD and suggests that the most requested gene FVL by physicians may not be sufficient to diagnose eventual future problems that can occur in the cardiovascular system. Subjects expressing the double mutations (Del/4G) are at high risk for the onset of CVDs.

Fig 1. Screening of Atherosclerosis and Venous Thrombosis-related gene’s mutations: allele’s frequencies.

DNAs were isolated from peripheral blood and transcribed and amplified by PCR as described in Materials and Methods; DNAs were from hypertensive hypercholesterolemic patients of the HH group (n = 144) versus Healthy group (n = 171). Relative distribution of mutated allele of genes related to Atherosclerosis (A) and Venous Thrombosis (B). Frequencies of mutated allele of the Angiotensin-converting Enzyme (ACE) (C) and Plasminogen Activator Inhibitor type 1 (PAI-1) (E) genes versus wild type. Polymorphism of ACE gene 287 bp insertion or deletion (Ins/Ins, Ins/Del and Del/Del) (D) and PAI-1 gene (5G/5G, 4G/5G and 4G/4G) (F). Results are expressed as percentages ± SEM of the number of patients detected with or without mutation from the total number of the studied population. Mutations are: ACE(Del), β-fibrinogen(-455G>A), HPA-1(b), ApoB(R3500Q) and ApoE(E4), blood coagulation factors II Prothrombin(G20210A), V(Leiden and R2) and XIII(V34L), the MTHFR(C677T and A1298C) and the PAI-1(4G).

Fig 2. Distribution of atherosclerosis and venous thrombosis-related genes in the healthy group (n = 171) between populations co-expressing allele “Del” of ACE and “4G” of PAI-1(Del/4G,+/+): Allele’s frequencies.

A, Relative distribution of atherosclerosis related polymorphisms. B, Relative distribution of venous thrombosis related genes. Results are expressed as percentages ± SEM of the number of patients detected with or without mutation from the number of the studied population. Co-expression Del/4G(+/+) (detected in 113 patients) or absence of Del/4G(-/-) (only 6 patients not expressing alleles Del/4G) (Total population, n = 171).

Fig 3. Distribution of risk factors that occur by gene’s mutations.

Co-expression of mutations by patient detected in total population of the healthy group (n = 171) or the hypertensive hypercholesterolemic group (n = 144). Results are expressed as percentages of from the total number of the studied population.

Fig 4. Evaluation of the number of risk factor to occur by patient between the Healthy and the HH groups.

Results are expressed as percentages of expressed gene’s mutation per (n) risk factor: 8(A), 7(B), 6(C), 5(D), 4(E) and 3(F). 12 polymorphisms are studied: Prothrombin FII(G20210A); FV-L(Leiden); FV-R2(R2); FXIII(V34L); MTHFR(C677T); MTHFR(A1298C); PAI-1(4G); ACE(Del); beta-fibrinogen FGB(-455G>A); HPA-1(b); ApoB(R3500Q); ApoE(E4).

Fig 5. Profile of cytokines secretion in cases co-expressing or not ACE/PAI-1(Del/4G).

Cytokines secretion in culture supernatants from peripheral blood mononuclear cells (PBMCs) obtained from the 6 healthy subjects not expressing the combined double risky alleles “Del” of ACE and “4G” of PAI-1 genes [Healthy-ACE/PAI-1(Del/4G)(-/-)], from 6 healthy subjects co-expressing Del/4G [Healthy-ACE/PAI-1(Del/4G)(+/+)] and from 6 patients HH co-expressing Del/4G [HH-ACE/PAI-1(Del/4G)(+/+)]. TNF-α (A), IFN-γ (B), IL-4 (C) and IL-10 (D) production were determined in triplicates (n = 24 for each group) and results are Mean ± SEM. *P<0.05 and **P<0.01; Del/4G(+/+) versus Del/4G(-/-).

Fig 6. Effects of ramipril (RAMP) and atorvastatin (ATV) on cytokines, ACE and PAI-1 secretion.

Culture supernatants from peripheral blood mononuclear cells (PBMCs) were obtained from the 6 healthy subjects not expressing the combined double risky alleles “Del” of ACE and “4G” of PAI-1 genes [Healthy-ACE/PAI-1(Del/4G)(-/-)], from 6 healthy subjects co-expressing Del/4G [Healthy-ACE/PAI-1(Del/4G)(+/+)] and from 6 patients HH co-expressing Del/4G [HH-ACE/PAI-1(Del/4G)(+/+)]. Cells were treated 48h with 10μM of RAMP and/or ATV. TNF-α (A), IFN-γ (B), IL-4 (C), IL-10 (D), ACE (E), PAI-1 (F) production were determined in triplicates (n = 24 for each group) and results are Mean ± SEM. ^§ P<0.05; Del/4G(+/+) versus Del/4G(-/-).*P<0.05 and **P<0.01; RAMP+ATV versus Control.