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Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3 -/- HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process "mitolipophagy" because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

UNC51-like kinase-1 (ULK1) is the catalytic component of the autophagy pre-initiation complex that stimulates autophagy via phosphorylation of ATG14, BECLN1 and other autophagy proteins. ULK1 has also been shown to specifically promote mitophagy but the mechanistic basis of how has remained unclear. Here we show that ULK1 phosphorylates the BNIP3 mitochondrial cargo receptor on a critical serine residue (S17) adjacent to its amino terminal LIR motif. ULK1 similarly phosphorylates BNIP3L on S35. Phosphorylation of BNIP3 on S17 by ULK1 promotes interaction with LC3 and mitophagy. ULK1 interaction also promotes BNIP3 protein stability by limiting its turnover at the proteasome. The ability of ULK1 to regulate BNIP3 protein stability depends on an intact "BH3" domain and deletion of its "BH3" domain reduces BNIP3 turnover and increases BNIP3 protein levels independent of ULK1. In summary ULK1 promotes mitophagy by both stabilization of BNIP3 protein and via phosphorylation of S17 to stimulate interaction with LC3., (© 2021. The Author(s).)

Breast cancer is the leading cause of cancer death among women worldwide. Blocking a single signaling pathway is often an ineffective therapy, especially in the case of aggressive or drug-resistant tumors. Since we have previously described the mechanism involved in the crosstalk between Retinoic Acid system and protein kinase C (PKC) pathway, the rationale of our study was to evaluate the effect of combining all-trans-retinoic acid (ATRA) with a classical PCK inhibitor (Gö6976) in preclinical settings. Employing hormone-independent mammary cancer models, Gö6976 and ATRA combined treatment induced a synergistic reduction in proliferative potential that correlated with an increased apoptosis and RARs modulation towards an anti-oncogenic profile. Combined treatment also impairs growth, self-renewal and clonogenicity potential of cancer stem cells and reduced tumor growth, metastatic spread and cancer stem cells frequency in vivo. An in-silico analysis of "Kaplan-Meier plotter" database indicated that low PKCα together with high RARα mRNA expression is a favorable prognosis factor for hormone-independent breast cancer patients. Here we demonstrate that a classical PKC inhibitor potentiates ATRA antitumor effects also targeting cancer stem cells growth, self-renewal and frequency.

Purpose: Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment., Methods: PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR., Results: Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARβ expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression., Conclusion: Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARβ receptor is required to ensure the effect on this process.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl 2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24 low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab., (© 2017 Wiley Periodicals, Inc.)

Purpose: We investigated the effects of laminin on the fraction of cells with self-renewing capacity in the estrogen-dependent, tamoxifen-sensitive LM05-E breast cancer cell line. We also determined whether laminin affected the response to tamoxifen., Materials and Methods: The LM05-E breast cancer cell line was used as a model for all experiments. Aldehyde dehydrogenase (ALDH) activity, clonogenic and mammosphere assays were performed to measure the effects of laminin on modulation of the stem cell subpopulation. Pluripotent gene expression was analyzed by reverse transcriptase-polymerase chain reaction. The involvement of the mitogen-activated protein kinase (MAPK)/ERK pathway was determined using specific inhibitors. The effects of laminin on the response to tamoxifenwere determined and the involvement of α6 integrin was investigated., Results: We found that pretreatment with laminin leads to a decrease in cells with the ability to form mammospheres that was accompanied by a decrease in ALDH activity. Moreover, exposure of mammospheres to laminin reduced the capacity to form secondary mammospheres and decreased the expression of Sox-2, Nanog, and Oct-4. We previously reported that 4-OH-tamoxifen leads to an increase in the expression of these genes in LM05-E cells. Treatment with signaling pathway inhibitors revealed that the MAPK/ERK pathway mediates the effects of laminin. Finally, laminin induced tamoxifen resistance in LM05-E cells through α6 integrin., Conclusion: Our results suggest that the final number of cells with self-renewing capacity in estrogen-dependent breast tumors may result from the combined effects of endocrine treatment and microenvironmental cues.

Background: Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment., Methods: The human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity., Results: We demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment., Conclusions: Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Traditional chemotherapies debulk tumors but fail to produce long-term clinical remissions due to their inability to eradicate tumor-initiating cells (TICs). This necessitates therapy with activity against the TIC niche. Αlpha6-integrin (α6-integrin) promotes TIC growth. In contrast, aryl hydrocarbon receptor (AhR) signaling activation impedes the formation of mammospheres (clusters of cells enriched for TICs). We investigated the ability of AhR agonist Aminoflavone (AF) and AF pro-drug (AFP464) to disrupt mammospheres derived from breast cancer cells and a M05 mammary mouse model of breast cancer respectively. We further examined the capacity of AF and AFP464 to exhibit anticancer activity and modulate the expression of 'stemness' genes including α6-integrin using immunofluorescence, flow cytometry and qRT-PCR analysis. AF disrupted mammospheres and prevented secondary mammosphere formation. In contrast, AF did not disrupt mammospheres derived from AhR ligand-unresponsive MCF-7 cells. AFP464 treatment suppressed M05 tumor growth and disrupted corresponding mammospheres. AF and AFP464 reduced the expression and percentage of cells that stained for 'stemness' markers including α6-integrin in vitro and in vivo respectively. These data suggest AFP464 thwarts bulk breast tumor and TIC growth via AhR agonist-mediated α6-integrin inhibition., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone-independent mammary cancer cell line LM38-LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38-LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic-vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self-renewal. Furthermore, Rottlerin pre-treatment induced in CSC the development of a "grape-like" morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self-renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self-renewal., (© 2015 Wiley Periodicals, Inc.)

It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all-trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA-induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone-independent breast cancer treatment., (© 2014 Wiley Periodicals, Inc.)

Purpose: Breast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model., Materials and Methods: The bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo., Results: We found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA., Conclusions: Our results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Using the M05 mouse mammary tumor model and the MCF-7 cell line, we investigated the effect of tamoxifen treatment on the fraction of breast cancer cells with self-renewing capacity both in vitro and in vivo. We found that pretreatment with 4-OH-tamoxifen leads to an increase in cells with the ability of forming mammospheres that express lower levels of ER-α and increased expression of transcription factors associated with pluripotency. Moreover, exposure on plastic to 4-OH-tamoxifen by itself leads to an upregulation of these transcription factors. M05 tumors grown in mice treated with tamoxifen have a higher percentage of cells with self-renewing capacity and this proportion is conserved when tumors are passaged to nontreated mice. Furthermore, interruption of tamoxifen leads to increased tumor growth compared to tumors grown in mice that were never exposed to the antiestrogen. In addition, these tumors are characterized by a higher number of CD24(l)CD29(h) cells compared to tumors grown in nontreated mice. Treatment in vitro with 4-OH-tamoxifen for 5 days leads to a long lasting increase in the proportion of cells with self-renewing capacity even after 1 month of growth in the absence of the antiestrogen. Finally, we compared the mammosphere forming capacity of hormone dependent and independent passages of the M05 tumor and found that hormone independence is associated to an increase in cells with self-renewing capacity. Our results support previous findings that suggest that endocrine treatment selects for cells with stem cell properties.

Doxorubicin is an anti-tumor antibiotic widely used in the management of cancer patients. Its main mechanism of action involves the generation of DNA damage and the inhibition of topoisomerase II, promoting apoptosis. AD 198 is a novel doxorubicin analog devoid of DNA binding and topoisomerase II inhibitory capacities. It has been proposed that AD 198 induces apoptosis by activating protein kinase C delta (PKCδ); a PKC isoform described as growth inhibitory in a large number of cell types. We have previously demonstrated that PKCδ overexpression in NMuMG cells induced the opposite effect, promoting proliferation and cell survival. In this study, we found that PKCδ overexpression confers an enhanced cell death resistance against AD 198 cytotoxic effect and against AD 288, another doxorubicin analog that preserves its mechanism of action. These resistances involve PKCδ-mediated activation of two well-known survival pathways: Akt and NF-κB. While the resistance against AD 198 could be abrogated upon the inhibition of either Akt or NF-κB pathways, only NF-κB inhibition could revert the resistance to AD 288. Altogether, our results indicate that PKCδ increases cell death resistance against different apoptosis inductors, independently of their mechanism of action, through a differential modulation of Akt and NF-κB pathways. Our study contributes to a better understanding of the mechanisms involved in PKCδ-induced resistance and may greatly impact in the rationale design of isozyme-specific PKC modulators as therapeutic agents.

Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings.

The migration of vascular smooth muscle cells (SMCs) and fibroblasts into the intima after vascular injury is a central process in vascular lesion formation. The elevation of transmural interstitial flow is also observed after damage to the vascular endothelium. We have previously shown that interstitial flow upregulates matrix metalloproteinase-1 (MMP-1) expression, which in turn promotes SMC and fibroblast migration in collagen I gels. In this study, we investigated further the mechanism of flow-induced MMP-1 expression. An ERK1/2 inhibitor PD-98059 completely abolished interstitial flow-induced SMC migration and MMP-1 expression. Interstitial flow promoted ERK1/2 phosphorylation, whereas PD-98059 abolished flow-induced activation. Silencing ERK1/2 completely abolished MMP-1 expression and SMC migration. In addition, interstitial flow increased the expression of activator protein-1 transcription factors (c-Jun and c-Fos), whereas PD-98059 attenuated flow-induced expression. Knocking down c-jun completely abolished flow-induced MMP-1 expression, whereas silencing c-fos did not affect MMP-1 expression. Taken together, our data indicate that interstitial flow induces MMP-1 expression and SMC migration in collagen I gels via an ERK1/2-dependent and c-Jun-mediated mechanism and suggest that interstitial flow, ERK1/2 MAPK, c-Jun, and MMP-1 may play important roles in SMC migration and neointima formation after vascular injury.

Complex hemodynamics play a role in the localization and development of atherosclerosis. Endothelial cells (ECs) lining blood vessel walls are directly influenced by various hemodynamic forces: simultaneous wall shear stress (WSS), normal stress, and circumferential stress/strain (CS) due to pulsatile flow, pressure, and diameter changes. ECs sense and transduce these forces into biomolecular responses that may affect intercellular junctions. In this study, a hemodynamic simulator was used to investigate the combined effects of WSS and CS on EC junctions with emphasis on the stress phase angle (SPA), the temporal phase difference between WSS and CS. Regions of the circulation with highly negative SPA, such as the coronary arteries and carotid bifurcation, are more susceptible to the development of atherosclerosis. At 5 h, expression of the tight junction protein zonula occludens-1 was significantly higher for the atheroprotective SPA = 0° compared to the atherogenic SPA = -180° while the apoptosis rate was significantly higher for SPA = -180° than SPA = 0°. This decrease in tight junction protein and increase in apoptosis and associated leaky junctions suggest a decreased junctional stability and a higher paracellular permeability for atherogenic macromolecules for the atherogenic SPA = -180° compared to SPA = 0°.

Although local wall shear stress (WSS) induced by blood flow has been implicated in atherogenesis, another prominent and often neglected hemodynamic feature, circumferential strain (CS) driven by pressure, is induced concurrently. To investigate endothelial cell (EC) responses to pathologic hemodynamics and their possible manipulation by pharmaceuticals, we simulated complete hemodynamic conditions comprised of simultaneous WSS and CS during treatment with conjugated linoleic acid (CLA), a known PPAR (-alpha and -gamma) activator and anti-atherogenic agent, on cultured EC and examined effects on gene and metabolite expression. Two hemodynamic conditions representative of distinct regions of the circulation, coronary arteries: pro-atherogenic (asynchronous WSS and CS) and straight descending aorta: non-atherogenic (synchronous WSS and CS), were applied to cultured EC during treatment with the nutraceutical CLA. Competitive-quantitative RT-PCR showed that asynchronous hemodynamics significantly reduced ( approximately 2-fold) eNOS and PPAR-gamma mRNA levels compared to synchronous hemodynamics at 5 and 12 h. ET-1 showed an opposite trend at 12 h. CLA treatment mitigated pro-atherogenic eNOS, ET-1, PPAR-alpha and -gamma mRNA expression profiles and NO and ET-1 secretion patterns during asynchronous hemodynamics. This study demonstrates the potential for a pharmacological treatment (CLA) to normalize pro-atherogenic gene expression profiles induced by hemodynamics inherent to the circulation.

Objective: Endothelium-derived vasoactive agents NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion. Complex hemodynamics characterized by the temporal phase angle between mechanical factors circumferential strain and wall shear stress (stress phase angle [SPA]) have been implicated in regions prone to pathologic development, such as atherosclerosis and intimal hyperplasia, in coronary and peripheral arteries where the mechanical forces are highly asynchronous (SPA=-180 degrees ). We determined the gene expression of endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2) affected by asynchronous hemodynamics (SPA=-180 degrees ) relative to normal hemodynamics (SPA=0 degrees ) in bovine aortic endothelial cells., Methods and Results: Quantitative competitive RT-PCR analysis showed that eNOS production (at 5 and 12 hours) and COX-2 production (at 5 hours) were reduced at the gene expression level by asynchronous hemodynamics (SPA=-180 degrees) compared with synchronous hemodynamics (SPA=0 degrees ), whereas ET-1 exhibited an opposite trend (at 5 and 12 hours). NO, ET-1, and PGI2 secretion followed their respective gene expression profiles after 5 and 12 hours., Conclusions: Together, these data suggest that highly asynchronous mechanical force patterns (SPA=-180 degrees ) can elicit proatherogenic vasoactive responses in endothelial cells at the gene expression level, indicating a novel mechanism that induces cardiovascular pathology.