Gender Differences in Physicians' Financial Ties to Industry: A Study of National Disclosure Data.

BACKGROUND: Academic literature extensively documents gender disparities in the medical profession with regard to salary, promotion, and government funded research. However, gender differences in the value of financial ties to industry have not been adequately studied despite industry's increasing contribution to income and research funding to physicians in the U.S. METHODS & FINDINGS: We analyzed publicly reported financial relationships among 747,603 physicians and 432 pharmaceutical, device and biomaterials companies. Demographic and payment information were analyzed using hierarchical regression models to determine if statistically significant gender differences exist in physician-industry interactions regarding financial ties, controlling for key covariates. In 2011, 432 biomedical companies made an excess of $17,991,000 in payments to 220,908 physicians. Of these physicians, 75.1% were male. Female physicians, on average, received fewer total dollars (-$3,598.63, p<0.001) per person than men. Additionally, female physicians received significantly lower amounts for meals (-$41.80, p<0.001), education (-$1,893.14, p<0.001), speaker fees (-$2,898.44, p<0.001), and sponsored research (-$15,049.62, p=0.05). For total dollars, an interaction between gender and institutional reputation was statistically significant, implying that the differences between women and men differed based on industry's preference for an institution, with larger differences at higher reputation institutions. CONCLUSIONS: Female physicians receive significantly lower compensation for similarly described activities than their male counterparts after controlling for key covariates. As regulations lead to increased transparency regarding these relationships, efforts to standardize compensation should be considered to promote equitable opportunities for all physicians.

The critical role of IL-1 receptor-associated kinase 4-mediated NF-κB activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis.

Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.

Loss of STAT1 is associated with increased aortic rupture in an experimental model of aortic dissection and aneurysm formation.

BACKGROUND: Transcription factor signal transducer and activator of transcription (STAT) 1 has been linked to a variety of pathologic states involved with matrix remodeling, but its role in aortic pathology has not been previously described. The current study hypothesized that STAT1 regulates aneurysmal degeneration and its role was evaluated in human abdominal aortic aneurysm (AAA) and in a mouse model of aortic dissection. METHODS: Apolipoprotein E knockout mice (ApoE-/-) or ApoE/STAT1 double knockout mice (ApoE/STAT1-/-) were infused with 1000 ng/kg/min of angiotensin II. Systolic blood pressure (SBP) was measured in the rodent tail. At sacrifice, aortic diameters and extent of aneurysm formation were measured by digital microscopy. STAT1 and phosphorylated-STAT1 protein levels were assessed in ApoE-/- mice at 0, 7, 14, and 28 days (n = 8/time point) by enzyme-linked immunosorbent assay. Histology was performed using hematoxylin and eosin (H&E) and Movat stains. Statistical analyses included chi(2) test, t test, and analysis of variance. RESULTS: STAT1 messenger RNA and total protein were greater in human AAA vs non-AAA controls. In addition, aneurysms occurred in 8%, 50%, and 80% of ApoE-/- mice at 7, 14, and 28 days, respectively. Total STAT1 levels were not altered during the course of angiotensin II infusion. Phosphorylated STAT1 levels peaked at 7 days with a 1.4-fold increase over baseline (P < .05). Aneurysms occurred in 0%, 100%, and 100% of ApoE/STAT1-/- mice at 3, 5, and 28 days. In mice infused with angiotensin II for >3 days, aortic rupture occurred more frequently in ApoE/STAT-/- mice (53% vs 19%, P < .05) and at earlier time points (4.0 +/- 0.5 vs 9.2 +/- 0.77 days, P < .05) vs ApoE-/- mice. SBP did not differ between the groups during angiotensin II infusion. By 28 days, aneurysms were larger in ApoE/STAT1-/- mice compared with ApoE-/- mice (2.7 +/- 0.4 vs 1.9 +/- 0.1 mm, P < .05) and were more extensive. H&E and Movat stain did not reveal differences in aortic wall structural content at baseline between ApoE-/- and ApoE/STAT1-/- mice. Both groups demonstrated equal disorganization in the aneurysmal state. CONCLUSIONS: Phosphorylated STAT1 is elevated during aneurysmal degeneration. Its loss is associated with a higher rate of acute aortic rupture and more extensive aneurysms in a mouse model of aortic dissection. Further investigation is necessary to determine whether these observations are secondary to an underlying aortic wall abnormality or alterations in vessel wall matrix remodeling.

Lysophosphatidylcholine activates a novel PKD2-mediated signaling pathway that controls monocyte migration.

OBJECTIVE: Monocyte activation and migration are crucial events in the development of atherosclerosis and other inflammatory diseases. This study examined the role of protein kinase D (PKD) in monocyte migration. Method and Results- PKD2 is the predominant isoform of PKD expressed in monocytic THP-1 cells and primary human monocytes. Lysophosphatidylcholine (lysoPC), a prominent component of oxidized low-density lipoprotein, induces rapid and marked PKD activation in these cells. Using multiple approaches, including dominant-negative mutants and small interfering RNA knock-down, we found that lysoPC-induced PKD2 activation was required for the activation of both ERK and p38 MAPK. p38 MAPK mediation of lysoPC-induced monocytic cell migration was reported previously; our results reveal that the lysoPC-induced PKD2-p38 pathway controls monocyte migration. CONCLUSIONS: This study provides the first evidence that (1) lysoPC activates PKD, (2) PKD2 has a novel role in p38 activation, and (3) the PKD2-activated p38 pathway is responsible for lysoPC-induced migration of THP-1 cells and human monocytes. Thus, PKD is a novel and functional intracellular regulator in both lysoPC signaling and monocyte migration. These results suggest a new role for PKD2 in the development of atherosclerosis and other inflammatory diseases.

Signal transducer and activator of transcription 1 is required for optimal foam cell formation and atherosclerotic lesion development.

BACKGROUND: Signal transducer and activator of transcription 1 (Stat1) potently regulates gene expression after stimulation by certain cytokines involved in tumorigenesis and host defenses. The present study investigated a novel role for Stat1 in foam cell formation and atherosclerosis. METHODS AND RESULTS: Inhibition of Stat1 activity by a Stat1-specific DNA "decoy" oligomer transfected into differentiated human THP-1 cells, and deficiency of stat1 in mouse macrophages significantly inhibited foam cell formation assessed by lipid staining and cholesteryl ester accumulation compared with control cells. The mechanism of Stat1 regulation of foam cell formation was uniquely dependent on the scavenger receptor CD36. Blunted Stat1 activity and stat1 deficiency significantly decreased expression of CD36 but not of scavenger receptor-A compared with controls, as assessed by immunoblotting and flow cytometry. Deficiency of CD36 but not scavenger receptor-A in mouse macrophages removed any dependency of foam cell formation on Stat1. In an intraperitoneal model of foam cell formation in which foam cells form in vivo independently of the model ligands used in vitro, stat1 deficiency significantly inhibited foam cell formation and CD36 expression. Transplantation of bone marrow from apolipoprotein e-/- x stat1-/- mice into lethally irradiated, atherosclerosis-susceptible apolipoprotein e-/- recipients significantly reduced both en face aortic lesion coverage and aortic root lesions compared with recipients of bone marrow from genetically matched apolipoprotein e-/- mice. CONCLUSIONS: Stat1 regulates CD36 expression and foam cell formation in macrophages in vitro; the Stat1 regulation of foam cell formation requires CD36. The regulation of CD36 expression by Stat1 may be important in other pathophysiological CD36-dependent events. Stat1 deficiency reduces atherosclerosis in an apolipoprotein e-/- atherosclerosis-susceptible bone marrow transplantation model.

Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines.

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFalpha, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFalpha induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the G(i)-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A(2) (PLA(2)) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.

Lysophosphatidic acid induction of tissue factor expression in aortic smooth muscle cells.

OBJECTIVE: Tissue factor (TF), the initiator of the coagulation cascade, is expressed by cells in atherosclerotic lesions. Lysophosphatidic acid (LPA) is a component of oxidized lipoproteins and an agent released by activated platelets. The present study investigated whether and how TF expression is regulated by LPA. METHODS AND RESULTS: Northern blotting, Western blotting, and TF activity assays demonstrated that LPA markedly induced TF mRNA, protein, and activity in vascular smooth muscle cells. LPA-induced TF expression is primarily controlled at the transcriptional level. Phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signaling-regulated kinases (ERK1/2) was rapidly and markedly induced by LPA. MEK inhibitors U0126 and PD98059 blocked both ERK activation and the increase in TF mRNA. In contrast, the specific p38 MAP kinase inhibitor SB203580 had no effect on LPA-induced TF mRNA increase. The Galpha(i) protein inhibitor, pertussis toxin, abolished LPA-induced phosphorylation of MEKs and ERKs, as well as the induction of TF mRNA. CONCLUSIONS: Our data demonstrate that a Galpha(i) protein and activation of MEKs and ERKs mediate LPA-induced TF expression. Our data suggest that elevated LPA could be a thrombogenic risk factor by upregulating TF expression. These results may have important implications in vascular remodeling and vascular diseases.

Smooth muscle cell proliferation induced by oxidized LDL-borne lysophosphatidylcholine. Evidence for FGF-2 release from cells not extracellular matrix.

Oxidized low-density lipoprotein (oxLDL), which accumulates in vascular lesions, alters vascular cell function in ways that can be construed as atherogenic. Among these is the observation that oxLDL and its lipids promote smooth muscle cell (SMC) proliferation. A number of schemes have been proposed to explain this phenomenon. Our published data support the concept that part of the proliferation is mediated by lysophosphatidylcholine (lysoPC) and structurally related phospholipids borne by oxLDL, which cause FGF-2 release via an oxidant-dependent mechanism. Since FGF-2 can bind extracellular matrices, we wanted to determine whether the FGF-2 released came from an intracellular or an extracellular matrix-bound pool. We tested whether lysoPC was capable of releasing FGF-2 from SMC matrices, whether agents that release FGF-2 from matrices could cause proliferation, and whether lysoPC-mediated proliferation could occur by stimulating metalloproteinase (MMP)-induced matrix degradation, which released matrix-bound FGF-2. Our results indicate that the source of FGF-2 released by lysoPC and related lipids is a preexisting cellular pool and not from matrix, and that the mechanism likely involves transient, sublethal cell permeabilization. These results enhance understanding of a mechanism by which oxLDL could contribute to SMC proliferation in arterial lesions.

The novel presenilin-1-associated protein is a proapoptotic mitochondrial protein.

Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimer's disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis.

Increased tissue factor expression predicts development of cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) limits the long-term success of cardiac transplantation. The incidence of CAV is increased in patients with elevated plasma levels of oxidized lipids or fibrin deposition within right heart biopsy (RHB) specimens. The present study investigated whether tissue factor (TF), the expression of which is regulated by oxidized lipids, is upregulated in patients with CAV. METHODS AND RESULTS: A TF score was developed to quantify TF expression in RHB specimens from 63 consecutive patients undergoing routine annual posttransplantation RHB and coronary angiography. In patients >2 years (3.0+/-0.8 years) posttransplantation (n=35), a high TF score was observed with greater frequency (75% versus 26%, P<0.004) in patients with CAV than those without CAV. In patients <2 years (0.87+/-0.48 years) posttransplantation (n=28) without evidence of CAV, the TF score was determined and patients were followed up prospectively. A high TF score had a positive predictive value of 78.6% for the development of CAV, and a low TF score had a negative predictive value of 100%. CONCLUSIONS: These data demonstrate that early TF expression predicts subsequent development of CAV. Increased TF expression could link the elevated levels of oxidized LDL and fibrin deposition known to precede CAV. These findings suggest that TF may play a role in the pathophysiology of CAV and could offer a potential prognostic tool and a novel target for the prevention of CAV, possibly with antioxidants or inhibitors of the TF pathway.

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis.

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.