Obesity is a growing health problem commonly associated with numerous metabolic disorders including type 2 diabetes, hypertension, cardiovascular disease, and some forms of cancer. The burden of obesity and associated cardiometabolic diseases are believed to arise through complex interplay between genetics and epigenetics predisposition, nutrition, environment, and lifestyle. However, the molecular basis and the repertoire of obesity-affecting factors are still unknown. Emerging evidence is connecting microRNAs (miRNAs) dysregulation with adipogenesis and obesity. Alteration in miRNAs expression could result in changes in the pattern of genes controlling a range of biological processes including inflammation, lipid metabolism, insulin resistance and adipogenesis. Hence, understanding exact roles of miRNAs as well as the degree of their contribution to the regulation of adipogenesis and fat cell development in obesity would provide new therapeutic targets for the development of novel and effective anti-obesity drugs. The objective of the current review is to: (i) discuss some of the latest development on relevant miRNAs dysregulation mainly in human adipogenesis and obesity, (ii) emphasize the role of circulating miRNAs as new promising therapeutics and attractive potential biomarkers for treating obesity and associated risk factor diseases, (iii) describe how dietary factors may influence obesity through modulation of miRNAs expression, (iv) highlight some of the actual limitations to the promise of miRNAs as novel therapeutics as well as to their translation for the benefit of patients, and finally (v) provide recommendations for future research on miRNA-based therapeutics that could lead to a breakthrough in the treatment of obesity and its associated pathologies.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The basic causes of CVD are not fully understood yet. Substantial evidence suggests that genetic predisposition plays a vital role in the physiopathology of this complex disease. Hence, identification of genetic contributors to CVD will likely add diagnostic accuracy and better prediction of an individual's risk. With high‐throughput genetics and genomics technology and newer genome‐wide study approaches, a number of genetic variations across the human genome were uncovered. Evidence suggests that genetic defects could influence CVD development and inter‐individual responses to widely used cardiovascular drugs like clopidogrel, aspirin, warfarin, and statins, and therefore, they may be integrated into clinical practice. If clinically validated, better understanding of these genetic variations may provide new opportunities for personalized diagnostic, pharmacogenetic‐based drug selection and best treatment in personalized medicine. However, numerous gaps remain unsolved due to the lack of underlying pathological mechanisms for how genetic predisposition could contribute to CVD. This review provides an overview of the extraordinary scientific progress in our understanding of genetic and genomic basis of CVD as well as the development of relevant genetic biomarkers for this disease. Some of the actual limitations to the promise of these markers and their translation for the benefit of patients will be discussed.

Abstract Context: Saponins have been reported to possess antitumor properties, to inhibit angiogenesis and to induce tumor apoptosis. Objective: To test the possible cytotoxic effect of crude extracts from four Caryophyllaceae species including Gypsophila paniculata L., Gypsophila trichotoma Wend., Saponaria officinalis L., and Dianthus sylvestris Wulffen on cultured monocyte/macrophage cell lines. Materials and methods: After acid hydrolysis of the methanol-aqueous extracts, two representative prosaponins of the Caryophyllaceae, gypsogenin 3-O-glucuronide and quillaic acid 3-O-glucuronide were purified using solid-phase extraction (SPE), then identified by ultra-performance liquid chromatography–electrospray/mass spectrometry (UPLC-ESI/MS). Cytotoxic activity of the crude extracts at concentrations ranging from 0.1 to 200 µg/ml was evaluated on rat alveolar macrophage NR8383 and human monocytic THP-1 cell lines. Apoptosis was determined by measuring caspase-3 activity. Results: Quantitative analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) revealed a high content of gypsogenin 3-O-glucuronide in Gypsophila species roots (0.52–1.13% dry weight). At a concentration ≥10 µg/ml of crude extracts, a significant reduction of NR8383 and THP-1 cell lines viability was evidenced using the Trypan blue exclusion test. D. sylvestris extract exhibited the highest toxicity against THP-1 cells. Caspase-3 activation was evidenced after 4 and 24 h incubation of macrophages with 100 µg/ml of S. officinalis and G. trichotoma extracts, indicating apoptosis induction. Discussion and conclusion: Crude extracts from the assayed species revealed cytotoxic effects toward macrophage cell lines. In Gypsophila species, gypsogenin 3-O-glucuronide derivatives could be responsible for the observed cytotoxicity. Therefore, crude extract of Caryophyllaceae is worth investigating for the potential development of agents against cancer cells.

The growing knowledge about genetic influence on cardiovascular diseases (CVD) combined with the recently generated amounts of genomic data hold promise to the identification of new markers for atherosclerotic CVD. Cardiovascular pharmacogenomics and pharmacogenetics have now the potential for leading to identification of genetic contributors and therefore to the development of predictive genetic tests that could optimize drugs efficacy and minimize toxicity. Clinical studies have shown that genetic variations within cytochromes P450 (CYPs), 3-Hydroxyl-3-Methylglutaryl Coenzyme A Reductase (HMGCR) and apolipoprotein E (APOE) genes influence individual's response to lipid lowering statins. Furthermore, development of antagonists or inhibitors of molecules such as peroxisome proliferator-activated receptors (PPARs), lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), angiotensin-converting enzyme (ACE), angiotensin receptors and tumor necrosis factor (TNF)-alpha could be another alternative to prevent atherosclerosis. In addition, novel molecules under the name of biologics including family of peptides such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), urocortin, apelin and antimicrobial peptides (AMPs) could be considered as new targets for the prevention and treatment of CVD. In this article, we will focus mainly on recent genomic advances in the development of new markers and therapeutic agents for CVD. We present an array of molecules that could have pharmacological benefit for the treatment of heart disease. We also discuss in details new strategies including biologics, which are actually the focus of companies for clinical development of therapeutic drugs. All these efforts provide optimism and attractive promise to cure CVD.

Background and aims: Antimicrobial peptides (AMPs) are components of the innate immune system. In addition, evidence suggests that these peptides are associated with various inflammatory diseases. We examined whether expression of the catheticidin LL-37 in peripheral blood mononuclear cells (PBMCs) is associated with cardiovascular risk factors.Methods and results: A total of 90 men and 87 women selected from STANISLAS cohort were studied. Expression of LL-37 mRNA isolated from PBMCs of these subjects was quantified by quantitative RTPCR. Anthropometric measurements and biochemical profiles were assessed for each individual. In women, LL-37 mRNA expression was significantly and positively correlated with body mass index (BMI) (p <= 0.001); waist circumference (WC) (p <= 0.01); systolic blood pressure (SBP) (p <= 0.05) and triglycerides (TG) level (p <= 0.05) and negatively with plasma levels of HDL-C (p <= 0.05). In men however, LL-37 was positively associated with waist to hip ratio (WHR) (p <= 0.05); SBP (p <= 0.901); TG (p <= 0.05); fasting glucose levels (p <= 0.01); alanine aminotransferase (ALT) activity (p <= 0.61); neutrophils counts (p <= 0.01) and negatively with Lymphocyte counts (p <= 0.001); serum HDL-C (p <= 0.001) and apoA-I (p <= 0.05) levels. After adjustment for WC and BMI, multiple regression analysis showed that LL-37 remained significantly associated with SBP; HDL-C; fasting glucose level; ALT activity; neutrophil and lymphocyte counts (p <= 0.001 to p <= 0.05) in men.Conclusion: Our results suggest that LL-37 gene expression may be closely associated with cardiovascular risk factors independently of BMI and WC. However, functional studies are required to confirm these data. (C) 2009 Published by Elsevier B.V.

Cathelicidin antimicrobial peptides are effectors of innate immune defense in mammals. Humans and mice have only one cathelicidin gene, whereas domesticated mammals such as the pig, cow, and horse have multiple cathelicidin genes. We hypothesized that the evolution of multiple cathelicidin genes provides these animals with enhanced resistance to infection. To test this, we investigated the effects of the addition of cathelicidins by combining synthetic cathelicidin peptides in vitro , by producing human keratinocytes that overexpress cathelicidins in culture, or by producing transgenic mice that constitutively overexpress cathelicidins in vivo . The porcine cathelicidin peptide PR-39 acted additively with human cathelicidin LL-37 to kill group A Streptococcus (GAS). Lentiviral delivery of PR-39 enhanced killing of GAS by human keratinocytes. Finally, transgenic mice expressing PR-39 under the influence of a K14 promoter showed increased resistance to GAS skin infection (50% smaller necrotic ulcers and 60% fewer surviving bacteria). Similarly constructed transgenic mice designed to overexpress their native cathelicidin did not show increased resistance. These findings demonstrate that targeted gene transfer of a xenobiotic cathelicidin confers resistance against infection and suggests the benefit of duplication and divergence in the evolution of antimicrobial peptides.

Hypertension is a multifactorial disorder that probably results from the inheritance of a number of susceptibility genes and involves multiple environmental determinants. Existing evidence suggests that the genetic contribution to blood pressure variation is about 30-50%. Although a number of candidate genes have been studied in different ethnic populations, results from genetic analysis are still inconsistent and specific causes of hypertension remain unclear. Furthermore, the abundance of data in the literature makes it difficult to piece together the puzzle of hypertension and to define candidate genes involved in the dynamic of blood pressure regulation. In this review, we attempt to highlight the genetic basis of hypertension pathogenesis, focusing on the most important existing genetic variations of candidate genes and their potential role in the development of this disease. Our objective is to review current knowledge and discuss limitations to clinical applications of genotypic information in the diagnosis, evaluation and treatment of hypertension. Finally, some principles of pharmacogenomics are presented here along with future perspectives of hypertension.

ABSTRACT Immune defense at an interface with the external environment reflects the functions of physical and chemical barriers provided by epithelial and immune cells. Resident epithelial cells, such as keratinocytes, produce numerous peptides with direct antimicrobial activity but also provide a physical barrier against invading pathogens and signal the recruitment of circulating immune cells, such as neutrophils. Antimicrobial peptides such as cathelicidin are produced constitutively by neutrophils and are inducible in keratinocytes in response to infection. The multiplicity of antimicrobial peptides and their cellular sources has resulted in an incomplete understanding of the role of cathelicidin production by epithelial cells in cutaneous immune defense. Therefore, this study sought to evaluate keratinocyte antimicrobial activity and the potential contribution of keratinocyte cathelicidin to host protection against two leading human skin pathogens. Wild-type mice and those with a targeted deletion of the cathelicidin gene, Cnlp , were rendered neutropenic prior to cutaneous infection. Interestingly, Cnlp -deficient mice remained more susceptible to group A streptococcus infection than mice with Cnlp intact, suggesting the involvement of epithelial cell-derived cathelicidin in host immune defense. Keratinocytes were then isolated in culture and found to inhibit the growth of Staphylococcus aureus , an effect that was partially dependent on their ability to synthesize and activate cathelicidin. Further, lentivirus-mediated delivery of activated human cathelicidin enhanced keratinocyte antimicrobial activity. Combined, these data illustrate the potential contribution of keratinocyte cathelicidin to the innate immune defense of skin against bacterial pathogens and highlight the need to consider epithelial antimicrobial function in the diagnosis and therapy of skin infection.

Cathelicidins are a class of small cationic peptide antibiotics that are expressed in skin and in other epithelial cells and are an active component of mammalian innate immunity. Human cathelicidin (hCAP18/LL-37) consists of a conserved prosequence called the cathelin-like domain and a C-terminal peptide named LL-37. To date, our understanding of the cathelin-like domain was very limited. To bring insight into the function of this evolutionarily conserved prosequence, we produced recombinant human cathelin-like protein and full-length hCAP18/LL-37 in Escherichia coli. As the cathelin-like protein shares homology with the cystatin family of cysteine protease inhibitors, we first analyzed the effect of the cathelin-like recombinant protein on the cysteine protease cathepsin L. We found that the cathelin-like protein inhibited protease activity. Next, we tested the cathelin-like protein for antimicrobial activity using solid phase radial diffusion and liquid phase killing assays. The cathelin-like prosequence, but not full-length hCAP18/LL-37, killed human pathogens including E. coli and methicillin-resistant Staphylococcus aureus at concentrations ranging from 16 to 32 microM. Together these findings suggest that after proteolytic cleavage the cathelin-like domain can contribute to innate host defense through inhibition of bacterial growth and limitation of cysteine-proteinase-mediated tissue damage. As these dual functions are complementary to the LL-37 peptide released from the C-terminus of full-length hCAP18/LL-37, human cathelicidin represents an elegant multifunctional effector molecule for innate immune defense of the skin.

Apolipoprotein (Apo) E is an important circulating and tissular protein involved in cholesterol homeostasis and many other functions. The common polymorphism in the coding region of the gene, four polymorphisms in the promoter region, other additional single nucleotide polymorphisms, as well as several ApoE variants have been identified. Outside genetic polymorphism effects, ApoE concentration is modulated in human plasma and tissue through many processes: 1) transcription regulation through hormone responsive elements; 2) cytokines; 3) compartmentalization in particles or linkage to HSPG; 4) degradation after oxidation, glycation, and proteolysis; and 5) through many specific and nonspecific receptor interactions. Is the level of ApoE in tissue or plasma critical in different pathologies such as cardiovascular diseases (CVD) or Alzheimer's disease (AD)? ApoE is able to bind to Abeta, tau, to be an antioxidant, to respond to inflammation, and is involved in cholesterol delivery, uptake, and accumulation. In experimental situations ApoE injection or positive modulation decreases cholesterol and triglycerides and improves cognitive impairment. ApoE peptides are involved in immune response. It seems more and more clear that low vs. high plasma concentration should be evaluated in large epidemiological studies. Only after such studies can the question be answered: Is a low or high concentration of ApoE beneficial or dangerous? This fascinating apolipoprotein will then be an interesting marker and/or drug target. (C) 2002 Wiley-Liss, Inc.

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4 < apoE3 < apoE2; T-max approximate to 52 < 56 < 59 degreesC, respectively). Helical contents at the onset and completion of denaturation (as monitored calorimetrically) were about 51% and 35%, respectively, for 22-kDa apoE3. As much as 70% of the unfolding enthalpy for 22-kDa apoE3 could be due to disruption of a-helix. The monomeric 22-kDa fragments unfold differently, with both apoE3 and apoE4 exhibiting unfolding intermediates, whereas apoE2 unfolds according to a two-state mechanism. Interactions with the C-terminal domain appear to destabilize the N-terminal domains in full-length apoE2 and apoE3, but less so for full-length apoE4. Self-association of full-length apoE at higher concentrations (1.5-2.5 mg/ml), as well as interaction of full-length apoE with phospholipid, enhances thermal stability. Cooperative changes in secondary structure, as monitored by circular dichroism, begin near 37 degreesC for full-length apoE3 and apoE4, indicating that full-length apoE3 and apoE4, but not apoE2, may be partially unfolded in vivo. The differences in stability and unfolding behavior are likely to contribute to the molecular basis for defects in lipid transport and neurological function induced by apoE polymorphism. (C) 2002 Elsevier Science B.V. All rights reserved.

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;-150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;-191) of apoE3 and apoE4 bound to the LDLR with approximately 100-fold greater affinity than the 22-kDa fragment of apoE2. The pK(a) values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pK(a) of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic alpha-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity. The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pK(a) of K146 significantly, but it increased the pK(a) of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1–191) in the lipid-free and lipid-associated states. As shown by 1H,13C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3·dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK a values above 10. In apoE3·DMPC complexes, however, all eight lysines were resolved, and the pK a values were 9.2–11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK a values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3·DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.