[Role of secreted and lipoprotein-associated phospholipase A2 in cardiovascular risk]. / Ruolo della fosfolipasi A2 secretoria e della fosfolipasi associata alle lipoproteine nel rischio cardiovascolare.

Phospholipase A(2) (PLA(2)) are enzymes that hydrolyze the ester bond of glycerophospholipids releasing free fatty acids and lysophospholipids, including the arachidonic acid, the precursor of the eicosanoids and the inflammatory cascades. PLA(2) are present in the atherosclerotic plaques and their direct involvement in the proatherogenic inflammatory response is well documented. Epidemiological and genetic studies have demonstrated the correlation of the PLA(2) mass and enzymatic activity with the incidence of cardiovascular diseases. The potential pro-atherogenic role of PLA(2) led to the development of two small molecules, varespladib, a reversible sPLA(2) inhibitor, and darapladib, a selective Lp-PLA(2) inhibitor. Both molecules have demonstrated antiatherosclerotic properties in animal models, and positive effects on atherosclerotic plaque composition evaluated in phase 2 clinical trials. On these grounds, the results of three phase 3 studies have recently been published: the VISTA-16 study with varespladib in patients with acute coronary syndrome, and the STABILITY and SOLID-TIMI 52 studies with darapladib in patients with stable coronary heart disease and acute coronary syndrome, respectively. Unexpectedly, both studies did not demonstrate an additional protective action of PLA 2 inhibitors over the standard of care treatment with statins, antiplatelet drugs, and coronary revascularization. In the present article, the enzymatic properties and the involvement of sPLA(2) and Lp-PLA(2) in atherogenesis are reviewed, with a focus on the results of experimental studies and clinical studies with both varespladib and darapladib inhibitors.

Specific roles for Group V secretory PLA2 in retinal iron-induced oxidative stress. Implications for age-related macular degeneration.

Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.

Associação da atividade da Lp-PLA2 e de antioxidantes lipossolúveis com marcadores cardiometabólicos em adolescentes / Association of Lp-PLA activity and fat-soluble antioxidants with cardiometabolic markers in adolescents

Introdução: A obesidade se caracteriza como um processo oxidativo e inflamatório, que predispõe adolescentes, de modo precoce, a eventos até recentemente pouco frequentes nessa faixa etária. Assim, a ação da enzima Fosfolipase A associada às lipoproteínas (Lp-PLA ), que reduz fosfolipídios oxidados e gera lisofosfolipídios, bem como a disponibilidade de antioxidantes plasmáticos, representam um importante tema de pesquisa no contexto cardiovascular. Objetivo: Verificar se a atividade da LP-PLA 2 2 e a concentração de antioxidantes lipossolúveis se associam com os principais marcadores de risco cardiovascular em adolescentes. Métodos: Duzentos e quarenta e dois adolescentes (10 a 19 anos), de ambos os sexos foram distribuídos, segundo o índice de massa corporal (IMC), em três grupos: Eutróficos (n=77), Sobrepeso (n=82) e Obesos (n=83). A amostra foi caracterizada através de parâmetros sócio-econômicos, estado de saúde, uso de medicamentos, antedecentes familiares de doenças crônicas e prática de atividade física. Foram avaliados ainda os dados antropométricos (peso, altura e composição corporal - bioimpedância), e o consumo alimentar por meio de três recordatórios 24 h. A partir de uma amostra de sangue coletada após jejum (12h), realizaram-se as análises da atividade da Lp-PLA , LDL(-) e seus auto-anticorpos, perfil lipídico (colesterol total, LDL-C, HDL-C e triglicerídeos), tamanho da HDL, proteína transportadora de éster de colesterol (CETP), ácidos graxos não esterificados (NEFAs), adipocitocinas, assim como antioxidantes (retinol, licopeno, -tocoferol e -caroteno) no plasma. Resultados: Artigo 1: Lp-PLA maybe an important cardiovascular biomarker in obese adolescents. Verificou-se que o perfil lipídico, insulina, HOMA-IR (resistência à insulina) e LDL(-) evidenciaram um maior risco cardiovascular nos adolescentes obesos. A atividade da enzima Lp-PLA 2 mostrou uma variação proporcional ao IMC, circunferência da cintura e porcentagem de gordura.

The role of lipoprotein-associated phospholipase A2 as a marker for atherosclerosis.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme that belongs to the superfamily of phospholipase A2 enzymes. Although initial studies showed that Lp-PLA2 might be protective against atherosclerosis, emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lysophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. This article reviews the potential mechanisms by which Lp-PLA2 may participate in the pathogenesis of atherosclerosis and its clinical manifestations, namely, coronary artery disease and stroke.

Chemotaxis: navigating by multiple signaling pathways.

During chemotaxis, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) accumulates at the leading edge of a eukaryotic cell, where it induces the formation of pseudopodia. PIP(3) has been suggested to be the compass of cells navigating in gradients of signaling molecules. Recent observations suggest that chemotaxis is more complex than previously anticipated. Complete inhibition of all PIP(3) signaling has little effect, and alternative pathways have been identified. In addition, selective pseudopod growth and retraction are more important in directing cell movement than is the place where new pseudopodia are formed.

Attenuated free cholesterol loading-induced apoptosis but preserved phospholipid composition of peritoneal macrophages from mice that do not express group VIA phospholipase A2.

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA(2)beta-null mice, and here we demonstrate that iPLA(2)beta-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA(2)beta-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA(2)betaexpression with recombinant adenovirus increases apoptosis toward WT levels. WT and iPLA(2)beta-null macrophages incorporate [(3)H]arachidonic acid ([(3)H]AA]) into glycerophosphocholine lipids equally rapidly and exhibit identical zymosan-induced, cPLA(2)alpha-catalyzed [(3)H]AA release. In contrast, although WT macrophages exhibit robust [(3)H]AA release upon FCL, this is attenuated in iPLA(2)beta-null macrophages and increases toward WT levels upon restoring iPLA(2)beta expression. Recent reports indicate that iPLA(2)beta modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA(2)beta-null cells. Immunoblotting studies indicate that iPLA(2)beta associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA(2)beta-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA(2)beta participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA(2)beta does not impair macrophage arachidonate incorporation or phospholipid composition.

Group V phospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages.

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A(2) (sPLA(2)-V) was required for COX-2 induction, but the nature of the sPLA(2)-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA(2)-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA(2)-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA(2)-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA(2)-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.

Essential role of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis.

Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca(2+). The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP(3) receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP(2) levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca(2+).

AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea.

Intracellular phospholipase A2 (PLA2) plays an important role in regulating oxylipin biosynthesis in mammals, but the molecular and biochemical nature of intracellular PLA2 is not well understood in plants. Arabidopsis thaliana gene At1g61850 (AtPLAI) encodes a 140-kDa protein that is most similar to mammalian calcium-independent PLA2, and additionally contains leucine-rich repeats and Armadillo repeats. AtPLAI hydrolyzes phospholipids at both the sn-1 and sn-2 positions, but prefers galactolipids to phospholipids as substrates. Profiling of lipid species altered in response to the necrotrophic fungus Botrytis cinerea revealed decreases in the levels of phosphatidylglycerol and digalactosyldiacylglycerol, suggesting that hydrolysis of plastidic polar lipids might provide precursors for pathogen-induced jasmonic acid (JA) production. Disruption of AtPLAI by T-DNA insertion reduced the basal level of JA, but did not impede pathogen-induced production of JA, free linolenic acid, or hydrolysis of plastidic lipids. Still, AtPLAI-deficient plants exhibited more damage than wild type plants after B. cinerea infection, and pretreatment of plants with methyl jasmonate alleviated pathogen damage to the mutant plants. The study shows that AtPLAI is an acyl hydrolase, rather than a specific phospholipase A. AtPLAI is involved in basal JA production and Arabidopsis resistance to the necrotrophic fungus B. cinerea.

Regulation of the NADPH oxidase activity and anti-microbial function of neutrophils by arachidonic acid.

Arachidonic acid (AA), a second-messenger molecule released from membrane phospholipids by phospholipase A(2) in activated cells, is a stimulator of neutrophil responses, including the oxygen-dependent respiratory burst. The polyunsaturated fatty acid is also the precursor of biologically active eicosanoids. There are several mechanisms by which AA stimulates the respiratory burst. These include the direct binding of AA to S100 proteins which regulate the assembly of the NADPH oxidase as well as the activation of key signaling molecules which control the respiratory burst. Arachidonic acid also stimulates it own release from membrane phospholipids and this contributes to optimal respiratory burst activity. Thus, increased levels of AA at sites of inflammation will influence the magnitude and course of the inflammatory response, not only by directly affecting the function of infiltrating neutrophils and other leukocytes, but also through its metabolites generated by lipoxygenases and cyclooxygenases.

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity.

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis.

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.

HCN pacemaker channel activation is controlled by acidic lipids downstream of diacylglycerol kinase and phospholipase A2.

Hyperpolarization-activated pacemaker currents (I(H)) contribute to the subthreshold properties of excitable cells and thereby influence behaviors such as synaptic integration and the appearance and frequency of intrinsic rhythmic activity. Accordingly, modulation of I(H) contributes to cellular plasticity. Although I(H) activation is regulated by a plethora of neurotransmitters, including some that act via phospholipase C (PLC), the only second messengers known to alter I(H) voltage dependence are cAMP, internal protons (H+(I)s), and phosphatidylinositol-4,5-phosphate. Here, we show that 4beta-phorbol-12-myristate-13-acetate (4betaPMA), a stereoselective C-1 diacylglycerol-binding site agonist, enhances voltage-dependent opening of wild-type and cAMP/H+(I)-uncoupled hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels, but does not alter gating of the plant hyperpolarization-activated channel, KAT1. Pharmacological analysis indicates that 4betaPMA exerts its effects on HCN gating via sequential activation of PKC and diacylglycerol kinase (DGK) coupled with upregulation of MAPK (mitogen-activated protein kinase) and phospholipase A2 (PLA2), but its action is independent of phosphoinositide kinase 3 (PI3K) and PI4K. Demonstration that both phosphatidic acid and arachidonic acid (AA) directly facilitate HCN gating suggests that these metabolites may serve as the messengers downstream of DGK and PLA2, respectively. 4BetaPMA-mediated suppression of the maximal HCN current likely arises from channel interaction with AA coupled with an enhanced membrane retrieval triggered by the same pathways that modulate channel gating. These results indicate that regulation of excitable cell behavior by neurotransmitter-mediated modulation of I(H) may be exerted via changes in three signaling lipids in addition to the allosteric actions of cAMP and H+(I)s.

Role of biomarkers in developing new therapies for vascular disease.

BACKGROUND: The use of biomarkers has been among the advances that have helped in the better medical management of patients. Biomarkers such as total cholesterol, low-density lipoprotein cholesterol, and hemoglobin A1c are used routinely. Advances in technologies, proteomics, and genomics and the resultant improved understanding of the pathophysiology of vascular diseases have led to the identification of several promising biomarkers. These biomarkers may be used to identify specific populations that may benefit from therapies, as surrogate markers of clinical efficacy, or as targets of therapy.

Differential behavior of sPLA2-V and sPLA2-X in human neutrophils.

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.

Combined activities of secretory phospholipases and eosinophil lysophospholipases induce pulmonary surfactant dysfunction by phospholipid hydrolysis.

BACKGROUND: Surfactant dysfunction is implicated in small airway closure in asthma. Increased activity of secretory phospholipase A(2) (sPLA(2)) in the airways is associated with asthma exacerbations. Phosphatidylcholine, the principal component of pulmonary surfactant that maintains small airway patency, is hydrolyzed by sPLA(2). The lysophosphatidylcholine product is the substrate for eosinophil lysophospholipases. OBJECTIVE: To determine whether surfactant phospholipid hydrolysis by the combined activities of sPLA(2)s and eosinophil lysophospholipases induces surfactant dysfunction. METHODS: The effect of these enzymes on surfactant function was determined by capillary surfactometry. Thin layer chromatography was used to correlate enzyme-induced changes in surfactant phospholipid composition and function. Phosphatidylcholine and its hydrolytic products were measured by using mass spectrometry. RESULTS: Eosinophils express a 25-kd lysophospholipase and group IIA sPLA(2). Phospholipase A(2) alone induced only a small decrease in surfactant function, and 25-kd lysophospholipase alone degraded lysophosphatidylcholine but had no effect on surfactant function. The combined actions of sPLA(2) and lysophospholipase produced dose-dependent and time-dependent losses of surfactant function, concomitant with hydrolysis of phosphatidylcholine and lysophosphatidylcholine. Lysates of AML14.3D10 eosinophils induced surfactant dysfunction, indicating these cells express all the necessary lipolytic activities. In contrast, lysates of blood eosinophils required exogenous phospholipase A(2) to induce maximal surfactant dysfunction. CONCLUSION: The combined activities of sPLA(2)s and eosinophil lysophospholipases are necessary to degrade surfactant phospholipids sufficiently to induce functional losses in surfactant activity as reported in asthma. CLINICAL IMPLICATIONS: The phospholipases and lysophospholipases expressed by eosinophils or other airway cells may represent novel therapeutic targets for blocking surfactant degradation, dysfunction, and peripheral airway closure in asthma.

An envelope-determined endocytic route of viral entry allows HIV-1 to escape from secreted phospholipase A2 entry blockade.

Secreted phospholipases A(2) (sPLA(2)s) represent a new class of human immunodeficiency virus (HIV) inhibitors that block the early steps of virus entry into cells. Here, we applied an in vitro evolution/selection procedure to select, from primary HIV isolates, an emerging variant (HIV(RBV-3)) able to actively infect cells in the presence of sPLA(2)s. HIV(RBV-3) represents a very atypical HIV-1 isolate because, in contrast to others, this virus requires a functional endocytic machinery to infect cells. Indeed, endocytosis inhibitors that affect endosome acidification (bafilomycin A(1), monensin) and/or endosomal trafficking (nocodazole, latrunculin A) drastically reduced HIV(RBV-3) replication. Using a standardized PCR-assay, we showed that endocytosis inhibitors block HIV(RBV-3) entry just before the reverse transcription step. Concurrently, to identify the viral proteins responsible for the HIV(RBV-3) atypical behaviour, we constructed a HIV-1 molecular chimera bearing different HIV(RBV-3) proteins. We demonstrated that the sole presence of the HIV(RBV-3) envelope glycoprotein is enough, not only to confer the resistance to sPLA(2)s, but also to direct HIV(RBV-3) to the endosomal-dependent entry pathway. Interestingly, HIV(RBV-3) envelope glycoprotein sequencing revealed an unusual structural pattern with the presence of rare mutations in the N-terminal region and V1-V2 envelope loop sequence extensions. Taken together, we conclude that HIV-1 may escape from entry inhibitors, such as sPLA2s, through the selection of a particular HIV-1 envelope glycoprotein that allows HIV to infect cells via an alternative entry route that relies on endosome trafficking.