Trends of vascular surgery procedures in Marfan syndrome and Ehlers-Danlos syndrome.

OBJECTIVES: Marfan syndrome and Ehlers-Danlos syndrome represent two connective tissue vascular diseases requiring unique consideration in their vascular surgical care. A comprehensive national review encompassing all hospitalizations for the Marfan Syndrome and Ehlers-Danlos syndrome patient population is lacking. METHODS: The National (Nationwide) Inpatient Sample from 2010 to 2014 was reviewed for all inpatient vascular surgery procedures including those with a diagnosis of Marfan syndrome and Ehlers-Danlos syndrome. National estimates of vascular surgery rates were generated from provided weights. Patient demographics, procedure type, and outcomes were assessed. RESULTS: There were 3103 Marfan syndrome and 476 Ehlers-Danlos syndrome vascular procedures identified as well as 3,895,381 vascular procedures in the remainder of population (control group). The percent of aortic procedures from all vascular procedures in Marfan syndrome (23.5%) and Ehlers-Danlos syndrome (23.5%) were 2.5-fold higher than controls (9.1%), p < 0.0001. Open aortic aneurysm repair was also significantly greater in both Marfan syndrome (16.8%) and Ehlers-Danlos syndrome (11.2%) compared to controls (4.4%), p < 0.0001. Endovascular aortic repair (p < 0.2302) was similar among the groups. Marfan syndrome (7.7%) and Ehlers-Danlos syndrome (5.1%) had more thoracic endovascular aortic repair performed than controls (0.7%), p < 0.0001. Percutaneous procedures were fewer in Marfan syndrome (6.3%) than controls (31.3%) and Ehlers-Danlos syndrome (26.3%), p < 0.0001, while repair of peripheral arteries was greater in Marfan syndrome (5.9%) and Ehlers-Danlos syndrome (4.1%) than controls (1.5%), p < 0.0001. For total aortic procedures, the mean age of aortic procedures was 68.2 years in controls vs 45.8 years in Marfan syndrome and 55.3 years in Ehlers-Danlos syndrome, p < 0.0001. Marfan syndrome and Ehlers-Danlos syndrome had fewer comorbidities overall, while controls had significantly higher rates of coronary artery disease (controls 39.9% vs Marfan syndrome 8.3% and Ehlers-Danlos syndrome 13.0%, p < 0.0001), peripheral vascular disease (controls 34.5% vs Marfan syndrome 4.2% and Ehlers-Danlos syndrome 8.7%, p < 0.0001), and diabetes (controls 20.6% vs Marfan syndrome 6.6 and Ehlers-Danlos syndrome 4.4%, p < 0.0001). Marfan syndrome and Ehlers-Danlos syndrome had higher overall complication rate (65.5% and 52.2%) compared to controls (44.6%), p < 0.0001. Postoperative hemorrhage was more likely in Marfan syndrome (42.9%) and Ehlers-Danlos syndrome (39.1%) than controls (22.2%), p < 0.0001. Increased respiratory failure was noted in Marfan syndrome (20.2%) vs controls (10.7%) and Ehlers-Danlos syndrome (8.7%), p = .0003. Finally, length of stay was increased in Marfan syndrome 12.5 days vs Ehlers-Danlos syndrome 7.4 days and controls 7.2 days (p < 0.0001) as well as a higher median costs of index hospitalization in Marfan syndrome ($57,084 vs Ehlers-Danlos syndrome $22,032 and controls $26,520, p < 0.0001). CONCLUSIONS: Patients with Marfan syndrome and Ehlers-Danlos syndrome differ from other patients undergoing vascular surgical procedures, with a significantly higher proportion of aortic procedures including open aneurysm repair and thoracic endovascular aortic repair. While they are younger with fewer comorbidities, due to the unique pathogenesis of their underlying connective tissue disorder, there is an overall higher rate of procedural complications and increased length of stay and cost for Marfan syndrome patients undergoing aortic surgery.

Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

Neurofibromin is a novel regulator of Ras-induced reactive oxygen species production in mice and humans.

Neurofibromatosis type 1 (NF1) predisposes individuals to early and debilitating cardiovascular disease. Loss of function mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, leads to accelerated p21(Ras) activity and phosphorylation of multiple downstream kinases, including Erk and Akt. Nf1 heterozygous (Nf1(+/-)) mice develop a robust neointima that mimics human disease. Monocytes/macrophages play a central role in NF1 arterial stenosis as Nf1 mutations in myeloid cells alone are sufficient to reproduce the enhanced neointima observed in Nf1(+/-) mice. Though the molecular mechanisms underlying NF1 arterial stenosis remain elusive, macrophages are important producers of reactive oxygen species (ROS) and Ras activity directly regulates ROS production. Here, we use compound mutant and lineage-restricted mice to demonstrate that Nf1(+/-) macrophages produce excessive ROS, which enhance Nf1(+/-) smooth muscle cell proliferation in vitro and in vivo. Further, use of a specific NADPH oxidase-2 inhibitor to limit ROS production prevents neointima formation in Nf1(+/-) mice. Finally, mononuclear cells from asymptomatic NF1 patients have increased oxidative DNA damage, an indicator of chronic exposure to oxidative stress. These data provide genetic and pharmacologic evidence that excessive exposure to oxidant species underlie NF1 arterial stenosis and provide a platform for designing novels therapies and interventions.

Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.

Neurofibromin-deficient myeloid cells are critical mediators of aneurysm formation in vivo.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity in circulating hematopoietic and vascular wall cells, which are critical for maintaining vessel wall homeostasis. NF1 patients have evidence of chronic inflammation resulting in the development of premature cardiovascular disease, including arterial aneurysms, which may manifest as sudden death. However, the molecular pathogenesis of NF1 aneurysm formation is unknown. METHOD AND RESULTS: With the use of an angiotensin II-induced aneurysm model, we demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) enhanced aneurysm formation with myeloid cell infiltration and increased oxidative stress in the vessel wall. Using lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1(+/-) aneurysm phenotype in vivo. Finally, oral administration of simvastatin or the antioxidant apocynin reduced aneurysm formation in Nf1(+/-) mice. CONCLUSION: These data provide genetic and pharmacological evidence that Nf1(+/-) myeloid cells are the cellular triggers for aneurysm formation in a novel model of NF1 vasculopathy and provide a potential therapeutic target.

Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.

Mutations in the NF1 tumor suppressor gene cause Neurofibromatosis type 1 (NF1). Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity. Some NF1 patients develop cardiovascular disease, which represents an underrecognized disease complication and contributes to excess morbidity and mortality. Specifically, NF1 patients develop arterial occlusion resulting in tissue ischemia and sudden death. Murine studies demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) in bone marrow cells enhances neointima formation following arterial injury. Macrophages infiltrate Nf1(+/-) neointimas, and NF1 patients have increased circulating inflammatory monocytes in their peripheral blood. Therefore, we tested the hypothesis that heterozygous inactivation of Nf1 in myeloid cells is sufficient for neointima formation. Specific ablation of a single copy of the Nf1 gene in myeloid cells alone mobilizes a discrete pro-inflammatory murine monocyte population via a cell autonomous and gene-dosage dependent mechanism. Furthermore, lineage-restricted heterozygous inactivation of Nf1 in myeloid cells is sufficient to reproduce the enhanced neointima formation observed in Nf1(+/-) mice when compared with wild-type controls, and homozygous inactivation of Nf1 in myeloid cells amplified the degree of arterial stenosis after arterial injury. Treatment of Nf1(+/-) mice with rosuvastatin, a stain with anti-inflammatory properties, significantly reduced neointima formation when compared with control. These studies identify neurofibromin-deficient myeloid cells as critical cellular effectors of Nf1(+/-) neointima formation and propose a potential therapeutic for NF1 cardiovascular disease.

Defective break-induced replication leads to half-crossovers in Saccharomyces cerevisiae.

Break-induced replication (BIR) is an important process of DNA metabolism that has been implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities, including loss of heterozygosity, translocations, and alternative telomere lengthening. Therefore, knowledge of how BIR is carried out and regulated is important for better understanding the maintenance of genomic stability in eukaryotes. Here we present a new yeast experimental system that enables the genetic control of BIR to be investigated. Analysis of mutations selected on the basis of their sensitivity to various DNA-damaging agents demonstrated that deletion of POL32, which encodes a third, nonessential subunit of polymerase delta, significantly reduced the efficiency of BIR, although some POL32-independent BIR was still observed. Importantly, the BIR defect in pol32Delta cells was associated with the formation of half-crossovers. We propose that these half-crossovers resulted from aberrant processing of BIR intermediates. Furthermore, we suggest that the half-crossovers observed in our system are analogous to nonreciprocal translocations (NRTs) described in mammalian tumor cells and, thus, our system could represent an opportunity to further study the NRT mechanism in yeast.

Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51.

DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.

Inverted DNA repeats channel repair of distant double-strand breaks into chromatid fusions and chromosomal rearrangements.

Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This "SSA-GCR" pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.