Citations

Background: The primary role of natriuretic peptide receptor 3 (NPR3) is clearance of natriuretic peptides. Genetic variation in NPR3 has been associated with blood pressure regulation and obesity. Despite its importance, variation in NPR3 characterizing coding single nucleotide polymorphisms (SNPs) has not been addressed by resequencing the gene. Objective: We set out to identify and functionally characterize common variation in NPR3 in 3 ethnic groups. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response. Also, rare variants identified by sequencing are enriched in complex disease traits. Methods and Results: DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3. There were 105 polymorphisms identified of which 50 were novel, including 8 nonsynonymous (ns) SNPs of which 7 were novel. Expression constructs for the nsSNPs were created and HEK-293 cells were transfected with constructs for wild type (WT) and the variants. Recombinant proteins were analyzed by quantitative Western blot analysis. A significant reduction in NPR3 immunoreactive protein (60% or less of WT) was observed for the following variants Cys3, Arg146, Val499, and Ser477+Asp521. The Arg146 novel polymorphism resulted in the least protein expression (20% of WT). After treatment of WT and Arg146 cells with proteasome (MG132) and autophagy (3MA) inhibtors, it was determined that autophagy and not proteasomes plays an important role in the degradation of the Arg 146 variant protein. Modeling based on the NPR3 x-ray crystallography structure confirmed that the Arg146 variant were not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: Multiple novel polymorphisms of NPR3 were identified in 3 ethnic groups. There were 4 variant receptors that displayed a significant decrease in NPR3 protein quantity, Arg146 being the variant with the least expression. Degradation of the variant Arg146 was mediated predominantly by autophagy. This SNP could have a significant effect on the metabolism of natriuretic peptides with clinical implications.

Introduction: Prior cardiac surgery can complicate the clinical presentation, diagnosis, and management of patients with type A aortic dissection (TAAAD). This report from the International Registry of Acute Aortic Dissection (IRAD) examines this hypothesis. Methods: 352 of 2289 TAAAD patients (15.4%) enrolled in IRAD had cardiac surgery prior to dissection, including coronary artery bypass grafting (CABG, 34.6%), aortic or mitral valve surgery (38.4%), aortic surgery (44.7%), and other cardiac surgery (18.1%). Results: Comparative differences in baseline demographics, clinical presentation, and management time are shown in Table 1. Patients with prior cardiac surgery were more likely to undergo CABG (p=0.006) or mitral valve replacement (p=0.001) at the time of dissection repair. Total cardiopulmonary bypass time was higher in patients with prior cardiac surgery (p<0.001); no difference was seen in cardiac arrest time (p=0.113) or cerebral ischemia time (p=0.286). In-hospital mortality was significantly higher for patients with prior cardiac surgery (33.5% vs. 24.0%, p70 (RR1.58, 95% CI 1.25-1.99), and medical management of acute dissection (RR 4.79, 95% CI 3.62-6.34). Kaplan-Meier analysis is shown in Figure 1. Conclusion: Prior cardiac surgery not only delays presentation and diagnosis of acute type A dissections, but is also an important adverse risk factor for early and late mortality.

Background: Stroke is one of the most dreaded complications of type A acute aortic dissection (TA-AAD). However, few data exist on its incidence and association with prognosis. Methods: We evaluated 2202 TA-AAD patients [pts, mean age 61.9 ± 14.4, 1487 (67.5%) male] from the International Registry of Acute Aortic Dissection (IRAD) to determine the incidence and prognostic influence of stroke in TA-AAD. Results: Stroke was present at arrival in 132 (6.0%) TA-AAD pts. Stroke pts were older (65±12 vs. 62±15 yrs; p=.002) and more likely to have hypertension (86% vs. 71%; p=.001) or atherosclerosis (29% vs. 22%; p=.042). While chest pain at arrival was less common (70% vs. 82%; p<.001), stroke pts presented more often with syncope (44% vs. 15%; p<.001), shock (14% vs. 7%; p=.005) or pulse deficit (51% vs. 29%; p=<.001). Arch vessels involvement was more frequent among the stroke pts (68% vs. 37%; p<.001). Stroke pts were treated less frequently by surgery (74% vs. 85%; p<.001). Hospital stay was significantly longer in patients presenting with stroke (median 17.9 versus 13.3 days, p<0.001). Stroke patients demonstrated more frequent in-hospital complications (Table) and higher mortality (adjusted OR 1.62, 95% CI .99-2.65, p=.055) Among hospital survivors, mortality at follow-up was similar in pts with and without stroke (adjusted HR 1.15, 95% CI 0.46-2.89, p=.761). Conclusions: Stroke occurred in greater than 1 of 20 pts with TA-AAD and was associated with increased morbidity and in-hospital, but not long-term mortality. Whether aggressive early intervention will reduce morbidity and improve mortality remains to be evaluated in future studies.

Background: In peripheral artery disease (PAD), claudication limits exercise performance, functional status and quality of life. Phosphodiesterase III inhibitors improve claudication-limited exercise performance in patients with PAD. K-134, a novel phosphodiesterase III inhibitor, was evaluated in a phase II trial incorporating an adaptive design to assess safety, quality of life and peak walking time (PWT) on a graded treadmill with cilostazol as an active comparator. Design: Patients with PAD were randomized to receive placebo (N = 87), K-134 at a dose of 25 mg (N = 42), 50 mg (N = 85) or 100 mg (N = 84), or cilostazol at a dose of 100 mg (N = 89), each given twice daily for 26 weeks. A Data Safety Monitoring Board (DSMB) implemented adaptive design was used to allow early discontinuation of unsafe or minimally informative K-134 arms. Interim safety assessments at 2 weeks included dosing of study medication at the study site followed by measurements of postural vital signs and symptoms, and at 4 weeks with a treadmill test at peak drug concentration to evaluate for ischemia or arrhythmias. Results: Based on prospectively defined adaptive criteria, the 25 mg K-134 arm was discontinued as the 50 mg and 100 mg doses were adequately tolerated, had no unanticipated safety signals, and were thus most likely to be informative concerning K-134 in the target population. In the intention-to-treat cohorts at 26 weeks PWT increased by 37% in the 100 mg K-134 arm and 23% on placebo (primary analysis p = 0.089). Using a mixed effects analysis in the entire population, 100 mg K-134 and cilostazol were each superior to placebo in improving PWT. In the per protocol population both K-134 100 mg and cilostazol 100 mg increased PWT as compared with placebo at 14 and 26 weeks. K-134 had tolerability and adverse effect profiles similar to that of cilostazol, with gastrointestinal adverse events, headache and dizziness the most common. Both drugs were associated with an increase in withdrawals prior to study completion due to adverse events as compared with placebo. Conclusions: K-134 was generally well tolerated, and at a dose of 100 mg twice daily had a safety profile and effect on treadmill walking performance that was similar to that of cilostazol.

Introduction: Creation of an atrial septal defect via atrial septostomy (AS) is a therapeutic option traditionally utilized for PAH in settings where medical therapy was unavailable. Its utility in the context of progressive disease despite medical therapy remains incompletely understood. Hypothesis: AS may be a useful option in patients with symptomatic PAH despite medical therapy and may improve functional class. Methods: Single center retrospective study of all adult PAH patients receiving AS at a tertiary PAH referral center. Results: Eleven atrial septostomy procedures were performed in 8 patients who were refractory to maximal medical management. All patients were WHO functional class III or IV and average patient age was 40 ± 13 years with a female predominance (89%). Mean RA pressure was 18 ± 4 mm Hg with cardiac index of 2.28 ± 0.82 L/min/m 2 prior to AS. Mean pulmonary artery pressure was 63 mm Hg with a pulmonary artery saturation of 48% and average systolic blood pressure of 97 mm Hg. Baseline NT-proBNP was 5573 ± 4589 pg/ml with a mean 6 minute walk distance (6MWD) of 311 ± 131 meters. Five of the 11 procedures resulted in functional class improvement. Functional class change did not relate to change in 6MWD or NT-pro BNP level. Three patients had repeat AS due to spontaneous narrowing of the original septostomy, with a mean duration of 220 ± 135 days between procedures. One patient died within 30 days of the procedure and another at day 88 while one patient received a heart/lung transplant at day 42 post AS. Mean follow up was 243 ± 183 days. The REVEAL risk scores ranged from 10 to 15, with predicted 1 year survival from pre-septostomy clinical data ranging from 22% to 80% (mean predicted survival of 57%). Actual transplant free survival at 1 year was 63%. Conclusions: Selected patients at high risk for 1 year mortality may receive symptomatic benefit from atrial septostomy. One year mortality remains high, and not significantly different from that predicted by the REVEAL score. Atrial septostomy may be a palliative treatment option for patients who are refractory to maximal medical therapy but further long term investigation is necessary.

Background: The tissue factor pathway regulates vascular structure in the adult through regulation of thrombin generation and signaling. Deletion of tissue factor pathway inhibitor (TFPI) in the mouse results in early intrauterine lethality, precluding study of vascular development. In addition, the carboxyl terminus of TFPI may have a TF-independent effect on vascular structure. We sought to determine the role of TFPI and its carboxyl terminus in vascular development of zebrafish. Methods and results: TFPI mRNA localization studies demonstrated expression in the developing kidney, gut, and vasculature of the zebrafish. We further assessed the role of TFPI in zebrafish angiogenesis, by injecting translational and splice-inhibiting morpholino (MO) oligonucleotides directed against zebrafish TFPI mRNA into embryos from the double transgenic zebrafish Tg(Fli1GFP); Tg(GATA1dsRed) expressing GFP in vascular endothelium and dsRed in erythroid cells. Knockdown of TFPI protein expression was subsequently confirmed by immunoblotting. In a dose-dependent manner, multiple non-overlapping MOs targeting the translation initiation site of TFPI independently and synergistically led to the formation of abnormally targeted intersegmental arteries which crossed somite boundaries by 48 hours post fertilization (hpf). Additionally, a plethoric caudal vein plexus with increased honeycombing was present by 24 hpf. This latter aspect of the phenotype was most striking after a MO targeted against a splice site involving the Kunitz 3 and carboxyl terminus domains of zebrafish TFPI was injected alone. Conclusions: TFPI is expressed during zebrafish development. Morpholino knockdown of TFPI expression alters both arterial and venous patterning in the developing zebrafish, indicating a fundamental role for TFPI in targeting of newly-formed blood vessels. Moreover, different domains of the TFPI protein may preferentially act on distinct vascular networks.

Introduction/Purpose: To examine trends in patient presentation, diagnosis and outcomes of acute aortic dissection over 15 years since the original report from the International Registry of Acute Aortic Dissection (IRAD). Methods: Patient data were collected at 28 international referral centers from 1996 up to the present. Trends were analyzed among 3275 patients divided according to date of enrollment into 6 equal groups and by Type: A (n=2110) or B (n=1165). Results: There was no change over time in the presenting complaints of severe or worst ever pain for Type A and Type B (90% and 96%, respectively), or in the incidence of chest pain ( 80% in Type A and 70% in Type B, respectively), with considerable overlap in chest vs. back pain between types. The report of pulse deficits did not change over time, present in 30% of Type A patients. Normal chest X-ray on presentation increased in Type A acute aortic dissection (AoD) from 13.7% to 31.3% over time (p-value for trend <0.001). In Type B the report of normal chest X-ray increased from 19.9% to 40.9% (p-value for trend <0.001). There were significant trends in initial imaging modalities for Type A: increased use of CT (from 49.4% to 71.6%, p-value for trend <0.001) and decreased use of TEE (from 45.3% to 25.4%, p-value for trend <0.001). For Type B, use of CT increased from 77.2% to 88.4% (p-value for trend .013). A decrease in overall mortality over time was significant for both Type A and Type B patients (see graph). Surgical mortality in Type A patients fell from 25.3% to 16.5% (p-value for trend 0 .003). Conclusions: No significant difference was seen over time in pain severity, chest pain, and presence of pulse deficits. The incidences of a normal chest X-ray, originally thought to be uncommon in dissection cases, increased over time to 30% in Type A and 40% in Type B AoD. Use of CT as the initial imaging modality increased for both Type A and Type B. Lastly, a significant decrease in overall mortality was seen for both Type A and Type B patients.

Background: Hematopoietic stem cells and the vasculature share a unique relationship during development, in the adult bone marrow and in the setting of extramedullary hematopoiesis within diseased calcified vessels. We hypothesized that the adult vasculature may contain progenitor cells with the capacity to reconstitute multi-lineage hematopoietic cells. Methods: Aortas from normal and ApoE −/− adult mice were disaggregated with collagenase and elastase. Disaggregates were cultured in methylcellulose for quantification of short and long term hematopoietic colony forming units (CFU). Flow cytometry assessed various populations of hematopoietic progenitor cells. Aortic disaggregates (or bone marrow or blood) from ROSA26 and GFP mice were transplanted into sublethally irradiated C57BL/6 recipients (n=6) via tail vein injections. Bone marrow cells from the primary recipients were transplanted at 4 months to secondary C57BL/6 recipients (n=6). Mice were sacrificed 3 months later. Blood and bone marrow were analyzed. Results: In short term culture in methylcellulose, aortic disaggregates generated erythroid, macrophage, granulocyte and megakaryocyte CFU, with predominance of macrophage colonies, primarily from adventitia (p<0.05) and a greater frequency was found in Apo E −/− mice (p<0.05). Long term culture defined that the combined frequency of the hematopoietic precursors approximates the frequency found in bone marrow (1:47,277). Flow cytometry of the aortic disaggregates indicates that among lin-/c-kit+/sca-1+ cells a small population of hematopoietic stem cells (CD150+endoglin+) and a population of multipotent progenitor cells (CD150− endoglin−) exist. Infusion of aortic disaggregates generated bone marrow, blood and spleen chimerism in primary and secondary transplanted mice at 3– 4 months. Flow cytometry of peripheral cells demonstrated multilineage hematopoietic cells. Conclusions: For the first time, we have identified a variety of progenitor cell populations in the aorta capable of hematopoiesis. Infusion of these cells into sublethally irradiated mice results in stable chimeras. These data support the presence of novel aortic hematopoietic progenitor cells.

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P < 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.