Methods and Results— ApoE^–/– mice were treated with TWEAK-inhibiting fusion protein, Fn14-Fc, in an early (5 to 17 weeks of age) or delayed (17 to 29 weeks of age) setting. In the aortic arch, Fn14-Fc as compared to control treatment resulted in advanced plaques which were smaller (early treatment), fewer (delayed treatment), lower in fibrotic content (early and delayed treatment), and exhibited an increased macrophage content and smaller macrophage size (delayed treatment). There were no differences in apoptosis in atherosclerotic plaques after Fn14-Fc versus control Ab treatment. However, blocking TWEAK resulted in less macrophage uptake of modified lipids in vitro.

Conclusions— Fn14-Fc fusion protein treatment did not prevent lesion initiation but inhibited some features of plaque progression and induced a unique advanced plaque phenotype with increased macrophage content and smaller macrophage size, which may be attributable to reduced lipid uptake. These findings indicate that TWEAK/Fn14 interactions regulate atherosclerosis and mediate lipid uptake in macrophages.

Methods and Results— LDLR-null mice receiving ritonavir were compared with those receiving ritonavir plus lipolysis inhibitor acipimox or vehicle alone to determine how acipimox would affect ritonavir-induced atherogenesis. Intermittent high-fat high-cholesterol diet was used to facilitate optimal atheromatous lesion development. Drug effects were assessed as changes in aortic lesion score, plasma lipid and lipoprotein profile, body fat mass, and insulin-induced suppression of plasma fatty acid concentrations. Ritonavir increased aortic lesions, in association with decreased body fat mass, impaired antilipolysis action of insulin, and increased proatherogenic plasma lipoproteins. All these adverse effects were attenuated by cotreatment with acipimox.

Conclusions— Our results provide the first direct evidence that supports the hypothesis that dysregulation of adipose lipolysis is an important contributor to the proatherogenic role of selected HIV protease inhibitors.

Methods and Results— Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1^{–/–ApoE–/–} mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1^{–/–ApoE–/–} mice analyzed by immunohistochemistry and echocardiography, respectively.

Conclusion— Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress–induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis.

Methods and Results— Cholesterol-rich diet feeding of miniature pigs was associated with an increase in PAFAH activity and an increase of the PAFAH to PON1 ratio. PLA2G7 RNA (coding for PAFAH) expression was increased in blood monocytes and plaque macrophages. Increased PAFAH activity was associated with higher plasma lysophosphatidylcholine and correlated with oxidized LDL. In THP1 monocytes and macrophages and in human blood-derived macrophages, oxidized LDL induced PLA2G7 RNA expression. Atherogenic diet feeding induced the accumulation of macrophages and oxidized LDL in the arterial wall leading to atherosclerosis. PAFAH activity correlated positively with plaque size and TNFalpha expression in plaque macrophages.

Conclusions— We demonstrated that an increase in PAFAH activity was associated with increased levels of lysophosphatidylcholine, oxidized LDL, and inflammation, resulting in accelerated atherosclerosis in hypercholesterolemic minipigs. The significant correlation between PLA2G7 RNA expression in plaque macrophages and plasma PAFAH activity suggests that the latter is a consequence, rather than a cause of macrophage accumulation. Our cell experiments suggest that oxidized LDL can induce PAFAH, resulting in accumulation of lysophosphatidylcholine that increases the inflammatory action of oxidized LDL.

Methods and Results— We generated human apoAII transgenic (Tg) rabbits, a species that normally does not have an endogenous apoAII gene. Plasma levels of human apoAII in Tg rabbits were 30 mg/dL, similar to the plasma levels in healthy humans. The expression of human apoAII in Tg rabbits resulted in increased levels of plasma triglycerides, total cholesterol, and phospholipids accompanied by a marked reduction in HDL-cholesterol levels compared with non-Tg littermates. Analysis of lipoprotein fractions showed that hyperlipidemia exhibited by Tg rabbits was caused by elevated levels of very-low-density lipoproteins (VLDL) and intermediate-density lipoproteins. Furthermore, postheparin lipoprotein lipase activity significantly decreased in Tg rabbits compared with non-Tg rabbits.

Conclusions— These results indicate that apoAII plays an important role in both VLDL and HDL metabolism, possibly through the inhibition of lipoprotein lipase activity. ApoAII Tg rabbits may become a new model for the study of human familial combined hyperlipidemia.

Methods and Results— ITX5061, a molecule initially characterized as a p38 MAPK inhibitor, increased HDL-C levels by 20% in a human population of hypertriglyceridemic subjects with low HDL levels. ITX5061 also moderately increased apoA-I but did not affect VLDL/LDL cholesterol or plasma triglyceride concentrations. ITX5061 increased HDL-C in WT and human apoA-I transgenic mice, and kinetic experiments showed that ITX5061 decreased the fractional catabolic rate of HDL-CE and reduced its hepatic uptake. In transfected cells, ITX5061 inhibited SR-BI–dependent uptake of HDL-CE. Moreover, ITX5061 failed to increase HDL-C levels in SR-BI^–/– mice. To assess effects on atherosclerosis, ITX5061 was given to atherogenic diet–fed Ldlr^+/– mice with or without CETP expression for 18 weeks. In both the control and CETP-expressing groups, ITX5061-treated mice displayed reductions of early atherosclerotic lesions in the aortic arch –40%, P<0.05), and a nonsignificant trend to reduced lesion area in the proximal aorta.

Conclusions— Our data indicate that ITX5061 increases HDL-C levels by inhibition of SR-BI activity. This suggests that pharmacological inhibition of SR-BI has the potential to raise HDL-C and apoA-I levels without adverse effects on VLDL/LDL cholesterol levels in humans.

Methods and Results— ApoE^–/– mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 µg/kg/d), anti-TWEAK neutralizing mAb (1000 µg/kg/d), TWEAK plus anti-TWEAK antibody (10 µg TWEAK +1000 µg anti-TWEAK/kg/d), or nonspecific IgG (1000 µg/kg/d) daily for 9 days. In ApoE^–/– mice, exogenous TWEAK administration in ApoE^–/– mice induced activation of NF-B, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE^–/– mice with an anti-TWEAK blocking mAb decreased NF-B activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion.

Conclusion— Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid–rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with hyperlipidemia.

Methods and Results— Mice were treated with once daily intraperitoneal injections of leptin (125 µg/mouse/d) for 2 months. The mice were then euthanized, and sections of the aortic root and thoracic aorta analyzed histomorphometrically. Measurements of lesion size and surface area occupied by atherosclerotic lesions did not reveal any differences between nontreated and leptin-treated animals. However, von Kossa staining of the aortic root demonstrated an 8.3±2.0-fold increase in lesion calcification as well as a 2.5±0.6-fold increase in valvular calcification in those animals treated with leptin. In addition, the percent total lesion area demonstrating ALP-positive staining was 5.4±2.1-fold greater in leptin-treated mice when compared to nontreated control mice. This increase in ALP staining was also accompanied by an increase in the expression of the osteoblast-specific markers, osteocalcin, and osteopontin.

Conclusions— Based on these observations, we conclude that leptin may increase cardiovascular risk by promoting osteogenic differentiation and thus vascular calcification.

Methods and Results— Chinese miniswine (n=28) were randomized to 1 of 4 groups (n=7 per group): control, SIMV (0.25 mg/kg · d), MSC transplantation, and SIMV+MSCs. AMI was created by ligating the left anterior descending coronary artery; MSCs were injected immediately into the cyanotic myocardium. At 6 weeks, MRI showed the number of dyskinetic segments and the infarct size were significantly decreased in the SIMV group. Cardiac function improved and the perfusion defect decreased significantly in the SIMV+MSC group but not in the MSC-only group (P<0.05, versus control group). MSC survival and differentiation were significantly better in the combination group than in the MSC-only group (P<0.01). Cell apoptosis decreased significantly in both the SIMV and the SIMV+MSC groups but not in the MSC-only group when compared with controls (P<0.05). Furthermore, oxidative stress and inflammatory response was significantly reduced in the infarcted regions in both the SIMV and the SIMV+MSCs groups.

Conclusions— SIMV treatment improves the therapeutic efficacy of MSC transplantation in acutely infarcted hearts by promoting cell survival and cardiovascular differentiation.

Methods and Results— Key parameters of cardiovascular function were compared among adult mice haploinsufficient for elastin (Eln^+/–), fibrillin-1 (Fbn1^+/–), or both proteins (dHet). Physiological and morphological comparisons correlate elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the elastin-deficient aorta.

Conclusions— These results demonstrate that elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.

Methods and Results— We showed a predominant arteriogenesis process in the thigh and a predominant angiogenesis-related process in the tibiofibular region, in response to ischemia during the first 15 days. After 15 days, mCT quantitative analysis reveals a remodeling of arterial neovessels and a regression depending on the restoration of the blood flow. We provided also new mCT data on the rapid and potent angiogenic effects of mesenchymal stem cell therapy on vessel formation and organization. We discussed the contribution of this technique compared with or in addition to data generated by the more conventional approaches.

Conclusion— This study demonstrated that optimized mCT is a robust method for providing new insights into the 3D understanding of postischemic vessel formation.

Methods and Results— qRT-PCR and Western blot confirmed highly increased FSS-dependent expression of Abra in growing collaterals. NO blockage by L-NAME abolished FSS-generated Abra expression as well as the whole arteriogenic process. Cell culture studies demonstrated an Abra-triggered proliferation of smooth muscle cells through a mechanism that requires Rho signaling. Local intracollateral adenoviral overexpression of Abra improved collateral conductance by 60% in rabbits compared to the natural response after FAL. In contrast, targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis.

Conclusions— FSS-induced Abra expression during arteriogenesis is triggered by NO and leads to stimulation of collateral growth by smooth muscle cell proliferation.

Methods and Results— To determine whether AngIV would inhibit fibrinolysis via PAI-1 activation and promote thrombosis, we evaluated the degree of fibrinolysis in thrombosis models and investigated the roles of AT4R after vascular injury using IRAP knockout mice (IRAP^–/–). In endothelial cells from control mice (WT; C57Bl6/J), both AngII and AngIV treatments increased PAI-1 mRNA expression in a dose-dependent manner, whereas the response was blunted in endothelial cells from IRAP^–/– mice. FeCl₃-induced thrombosis was suppressed in the carotid arteries of IRAP^–/– mice when compared with WT mice. Similarly, in a model of carotid artery ligation and cuff placement, IRAP^–/– mice demonstrated accelerated fibrinolysis 7 days after surgery and reduced occlusive thrombosis with negative remodeling at 28 days.

Conclusions— AngIV-AT4R signaling has a key role in fibrinolysis and the subsequent formation of arterial thrombosis after vascular injury. AT4R may be a novel therapeutic target against cardiovascular disease.

Methods and Results— Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)–dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti–PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1–deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation.

Conclusion— Thrombin induces MMP-10 through a PAR-1–dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

Methods and Results— The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo.

Conclusion— The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner.

Methods and Results— When murine ES cell-derived VEGFR2⁺ cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458) and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress-induced NICD translocation, and that, in turn, blocked the shear stress-induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression.

Conclusion— Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.

Methods and Results— Northern blot analysis and ELISA revealed that LPS-induced TNF- upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF- upregulation in HIF-1–depleted cells and HIF-1 overexpression failed to suppress the induction of TNF-. DMOG rather suppressed LPS-induced NF-B transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF- induction. DMOG also inhibited LPS-induced TNF- production in peritoneal macrophages as well as human macrophages.

Conclusions— PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF- upregulation in macrophages possibly through NF-B inhibition, which is independent of HIF-1 accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases.

Methods and Results— In THP-1 monocytes treated with TNF- (1 to 100 pmol/L/30 minutes), cytosolic RhoA small GTPase rapidly translocated to the plasma membrane via functionally active ezrin/radixin/moesin (ERM) complex, a cytoskeletal linker, and subsequent actin polymerization through NF-B activation. The threonine phosphorylation of ERM was accomplished by the activation of TNF receptor type I (TNFRI) and signaling pathways involving PI3K and an atypical PKC; ie, PKC. The TNF--treated monocytes (10 pmol/L) displayed more potent and prolonged generation of GTP-bound RhoA in response to secondary stimulation with RhoA-activating monocyte chemoattractant protein-1 (MCP-1). Clearly, human circulating monocytes preconditioned by 10 pmol/L TNF- augmented MCP-1–mediated chemotaxis and firm adhesion on VCAM-1 and ICAM-1 in vitro and ex vivo. The elevation of serum TNF- (>5 pmol/L within 16 hours), which was introduced by intraperitoneal injection of mouse-specific TNF- to C57/BL6 mice, enhanced the number of CD80+ monocytes transmigrating to the JE/MCP-1–injected intraperitoneal space.

Conclusions— Picomolar concentrations of TNF- in the bloodstream may prime the RhoA-dependent activities of circulating monocytes to enhance recruitment to active inflammatory foci.

Methods and Results— Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red–labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-B in HCAECs.

Conclusions— Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

Methods and Results— By screening phosphorylation-deficient and mimetic mutations in SRF^–/– embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG.

Conclusions— Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.

Methods and Results— Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167, and threonine (T) 231 in HEK293 cells, whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51, S72, T85, T91, T103, S130, S167 and T231). GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.

Conclusion— Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103, and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.

Methods and Results— Formation of redox-active phosphatidylcholine hydroperoxides (PCOOH) and redox-inactive phosphatidylcholine hydroxides (PCOH) was initiated in LDL by free radical-induced oxidation. Human HDL3 inactivated LDL-derived PCOOH (–62%, P<0.01) and enhanced accumulation of PCOH (2.1-fold, P<0.05); in parallel, HDL3 accumulated minor amounts of PCOOH. Enzyme-deficient reconstituted dense HDL potently inactivated PCOOH (–43%, P<0.01). HDL3-mediated reduction of PCOOH to PCOH occurred concomitantly with oxidation of methionine residues in HDL3-apolipoprotein AI (apoAI). Preoxidation of methionine residues by chloramine T markedly attenuated PCOOH inactivation (–35%); by contrast, inhibition of HDL3-associated enzymes was without effect. PCOOH transfer rates from oxidized LDL to phospholipid liposomes progressively decreased with increment in the rigidity of the phospholipid monolayer.

Conclusions— The redox status of apoAI and surface lipid rigidity represent major determinants of the potent HDL3-mediated protection of LDL against free radical-induced oxidation. Initial transfer of PCOOH to HDL3 is modulated by the surface rigidity of HDL3 particles with subsequent reduction of PCOOH to PCOH by methionine residues of apoAI.

Methods and Results— The prospective observational Cancer and Thrombosis Study (CATS) includes patients with newly diagnosed cancer or disease progression, study end point is symptomatic VTE. FVIII was measured on a Sysmex CA 7000 analyzer. Data on 840 patients (median age: 62 years, 25th to 75th percentile 53 to 68, 378 women) were available for analyses, of these 111 patients had hematologic malignancies and 729 solid cancer. During a median observation time of 495 days 62 events occurred. Cumulative probability of VTE after 6 months was 14% in patients with elevated FVIII-levels and 4% in those with normal levels (P=0.001). The association was strongest in younger patients: whereas in 40-year-old patients a 2-fold VTE risk per factor VIII increase of 20% was observed (HR=2.0 [95% CI: 1.5 to 2.7], P<0.0001), this association was still present but attenuated in older patients.

Conclusions— FVIII is independently associated with an increased risk of VTE in cancer patients. The association between FVIII and VTE risk declines with increasing age.

Methods and Results— Cardiac death and nonfatal MI events were identified in the ARIC cohort during follow-up from 1987 through 2001. Markers of inflammation and hemostasis were determined at baseline using standardized procedures. Cox proportional hazard regression and polytomous logistic regression were used to estimate associations. We observed a positive gradient in incidence of sudden cardiac death (SCD), nonsudden cardiac death (NSCD), and nonfatal MI in association with decreasing levels of albumin and increasing levels of white blood cell count and of markers of hemostasis (fibrinogen, von Willebrand factor, factor VIIIc). Associations for von Willebrand factor were stronger for fatal relative to nonfatal events (3rd versus 1st tertile hazard ratios: SCD 3.11 [95% CI 2.10, 4.59], NSCD 2.12 [95% CI 1.28, 3.49], nonfatal MI 1.42 [95% CI 1.19, 1.70]). For factor VIIIc those associations were strongest for sudden cardiac death: SCD 3.16 (95% CI 2.18, 4.58), NSCD 1.44 (95% CI 0.93, 2.24), nonfatal MI 1.54 (95% CI 1.29, 1.84). Gradients of association for fibrinogen and white blood cell count, examined over tertiles of distribution and per one SD increase, were similar for the 3 end points. All associations were independent of smoking status.

Conclusion— von Willebrand factor and factor VIIIc are associated with an increased risk of cardiac death as compared to the risk of nonfatal MI.

Methods and Results— A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, 50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9.

Conclusion— These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL.