Intraoperative echocardiographic contrast opacifies the left atrial appendage and assists in surgical exclusion.

Surgical exclusion of the left atrial appendage (LAA) for stroke prevention in atrial fibrillation is frequently incomplete and remains to be optimized. We present a man who did not tolerate anticoagulant and failed percutaneous occlusion. Intraoperative echocardiographic contrast was used to assist surgical exclusion. Follow-up showed a persistent occlusion.

Prothrombin complex concentrates in cardiac surgery: where are we?

PURPOSE OF REVIEW: Major bleeding in cardiac surgery is commonly encountered, and, until recently, most frequently managed with fresh frozen plasma (FFP). However, a Cochrane review found this practice to be associated with a significant increase in red blood cell (RBC) transfusions and costs. These findings have led to off-label uses of prothrombin complex concentrates (PCCs) in cardiac surgery. The purpose of this review is to compare and contrast the use of FFP and PCC, review the components, limitations and risks of different types of PCCs, and discuss the latest evidence for the use of PCC versus FFP in cardiac surgery. RECENT FINDINGS: A recent review and meta-analysis suggests that PCC administration in cardiac surgery is more effective than FFP in reducing RBC transfusions and costs. SUMMARY: The current data supports the use of 4F-PCC instead of FFP as the primary hemostatic agent in cases of major bleeding in cardiac surgery. The use of PCCs is associated with reduced rates of RBC transfusions while maintaining a favorable safety profile. Clear advantages of PCC over FFP include its smaller volume, higher concentration of coagulation factors and shorter acquisition and administration times.

Stimulation of Caveolin-1 Signaling Improves Arteriovenous Fistula Patency.

Objective- Arteriovenous fistulae (AVF) are the most common access created for hemodialysis; however, many AVF fail to mature and require repeated intervention, suggesting a need to improve AVF maturation. Eph-B4 (ephrin type-B receptor 4) is the embryonic venous determinant that is functional in adult veins and can regulate AVF maturation. Cav-1 (caveolin-1) is the major scaffolding protein of caveolae-a distinct microdomain that serves as a mechanosensor at the endothelial cell membrane. We hypothesized that Cav-1 function is critical for Eph-B4-mediated AVF maturation. Approach and Results- In a mouse aortocaval fistula model, both Cav-1 mRNA and protein were increased in the AVF compared with control veins. Cav-1 KO (knockout) mice showed increased fistula wall thickening ( P=0.0005) and outward remodeling ( P<0.0001), with increased eNOS (endothelial NO synthase) activity compared with WT (wild type) mice. Ephrin-B2/Fc inhibited AVF outward remodeling in WT mice but not in Cav-1 KO mice and was maintained in Cav-1 RC (Cav-1 endothelial reconstituted) mice (WT, P=0.0001; Cav-1 KO, P=0.7552; Cav-1 RC, P=0.0002). Cavtratin-a Cav-1 scaffolding domain peptide-decreased AVF wall thickness in WT mice and in Eph-B4 het mice compared with vehicle alone (WT, P=0.0235; Eph-B4 het, P=0.0431); cavtratin also increased AVF patency (day 42) in WT mice ( P=0.0275). Conclusions- Endothelial Cav-1 mediates Eph-B4-mediated AVF maturation. The Eph-B4-Cav-1 axis regulates adaptive remodeling during venous adaptation to the fistula environment. Manipulation of Cav-1 function may be a translational strategy to enhance AVF patency.

CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues.

Circadian locomotor output cycles kaput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo, ClockS845A knockin mice were generated using CRISPR/Cas9 technology. ClockS845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that ClockS845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1, Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues.

Eph-B4 regulates adaptive venous remodeling to improve arteriovenous fistula patency.

Low rates of arteriovenous fistula (AVF) maturation prevent optimal fistula use for hemodialysis; however, the mechanism of venous remodeling in the fistula environment is not well understood. We hypothesized that the embryonic venous determinant Eph-B4 mediates AVF maturation. In human AVF and a mouse aortocaval fistula model, Eph-B4 protein expression increased in the fistula vein; expression of the arterial determinant Ephrin-B2 also increased. Stimulation of Eph-B-mediated signaling with Ephrin-B2/Fc showed improved fistula patency with less wall thickness. Mutagenesis studies showed that tyrosine-774 is critical for Eph-B4 signaling and administration of inactive Eph-B4-Y774F increased fistula wall thickness. Akt1 expression also increased in AVF; Akt1 knockout mice showed reduced fistula diameter and wall thickness. In Akt1 knockout mice, stimulation of Eph-B signaling with Ephrin-B2/Fc showed no effect on remodeling. These results show that AVF maturation is associated with acquisition of dual arteriovenous identity; increased Eph-B activity improves AVF patency. Inhibition of Akt1 function abolishes Eph-B-mediated venous remodeling suggesting that Eph-B4 regulates AVF venous adaptation through an Akt1-mediated mechanism.

Improving the Outcome of Vein Grafts: Should Vascular Surgeons Turn Veins into Arteries?

Autogenous vein grafts remain the gold standard conduit for arterial bypass, particularly for the treatment of critical limb ischemia. Vein graft adaptation to the arterial environment, i.e., adequate dilation and wall thickening, contributes to the superior performance of vein grafts. However, abnormal venous wall remodeling with excessive neointimal hyperplasia commonly causes vein graft failure. Since the PREVENT trials failed to improve vein graft outcomes, new strategies focus on the adaptive response of the venous endothelial cells to the post-surgical arterial environment. Eph-B4, the determinant of venous endothelium during embryonic development, remains expressed and functional in adult venous tissue. After surgery, vein grafts lose their venous identity, with loss of Eph-B4 expression; however, arterial identity is not gained, consistent with loss of all vessel identity. In mouse vein grafts, stimulation of venous Eph-B4 signaling promotes retention of venous identity in endothelial cells and is associated with vein graft walls that are not thickened. Eph-B4 regulates downstream signaling pathways of relevance to vascular biology, including caveolin-1, Akt, and endothelial nitric oxide synthase (eNOS). Regulation of the Eph-B4 signaling pathway may be a novel therapeutic target to prevent vein graft failure.

Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression.

The AKT signaling pathway is important for circadian rhythms in mammals and flies ( Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1-/- mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1-/- aorta, compared with control aorta, follows a distinct pattern. In the Akt1-/- aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms.

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase.

OBJECTIVE: Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be through regulation of downstream eNOS activity. METHODS: Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type or eNOS knockout mice. RESULTS: Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release (n = 3; P < .01) as well as increased endothelial cell migration (n = 6; P < .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n = 7; P < .05). CONCLUSIONS: eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.

Pericardial patch venoplasty heals via attraction of venous progenitor cells.

Pericardial patches are commonly used during cardiovascular surgery to close blood vessels. In arteries, patches accumulate arterial progenitor cells; we hypothesized that venous patches would accumulate venous progenitor cells, in the absence of arterial pressure. We developed a novel rat inferior vena cava (IVC) venotomy model and repaired it with a pericardial patch. Cells infiltrated the patch to form a thick neointima by day 7; some cells were CD34(+)/VEGFR2(+) and CD31(+)/Eph-B4(+) consistent with development of venous identity in the healing patch. Compared to arterial patches, the venous patches had increased neointimal thickness at day 7 without any pseudoaneurysms. Addition of an arteriovenous fistula (AVF) to increase blood flow on the patch resulted in reduced patch neointimal thickness and proliferation, but neointimal thickness was not reversible with AVF ligation. These results show that rat patch venoplasty is a novel model of aggressive venous neointimal hyperplasia.

Membrane-mediated regulation of vascular identity.

Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane-associated molecules, particularly the Eph-ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane-associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell-to-cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph- and ephrin-related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane-associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane-bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph- and ephrin-related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies.

Future research directions to improve fistula maturation and reduce access failure.

With the increasing prevalence of end-stage renal disease, there is a growing need for hemodialysis. Arteriovenous fistulae (AVF) are the preferred type of vascular access for hemodialysis, but maturation and failure continue to present significant barriers to successful fistula use. AVF maturation integrates outward remodeling with vessel wall thickening in response to drastic hemodynamic changes in the setting of uremia, systemic inflammation, oxidative stress, and pre-existent vascular pathology. AVF can fail due to both failure to mature adequately to support hemodialysis and development of neointimal hyperplasia that narrows the AVF lumen, typically near the fistula anastomosis. Failure due to neointimal hyperplasia involves vascular cell activation and migration and extracellular matrix remodeling with complex interactions of growth factors, adhesion molecules, inflammatory mediators, and chemokines, all of which result in maladaptive remodeling. Different strategies have been proposed to prevent and treat AVF failure based on current understanding of the modes and pathology of access failure; these approaches range from appropriate patient selection and use of alternative surgical strategies for fistula creation, to the use of novel interventional techniques or drugs to treat failing fistulae. Effective treatments to prevent or treat AVF failure require a multidisciplinary approach involving nephrologists, vascular surgeons, and interventional radiologists, careful patient selection, and the use of tailored systemic or localized interventions to improve patient-specific outcomes. This review provides contemporary information on the underlying mechanisms of AVF maturation and failure and discusses the broad spectrum of options that can be tailored for specific therapy.

Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.

The National Cancer Institute best case series program: a summary of cases of cancer patients treated with unconventional therapies in India.

OBJECTIVES: The National Cancer Institute (NCI) Best Case Series (BCS) Program provides an independent review of medical records, imaging, and pathology of cancer patients treated with unconventional therapies. The goal of the NCI BCS Program is to identify preliminary evidence of tumor regression and assess whether there is sufficient evidence to move forward with NCI-initiated research. The objective was to review case reports submitted by 4 practitioners from India who used ayurvedic and homeopathic therapies to treat cancer. DESIGN: Retrospective review of case reports of 4 practitioners from India who used ayurvedic and homeopathic therapies to treat cancer. RESULTS: A total of 68 cases were submitted to the NCI BCS Program. Fifty-one percent of the cases represented homeopathy and 49% ayurveda. Of the 68 cases, 32 (47%) of the cases were collectively designated as "persuasive" (P) or "supportive"(S), and 36 (53%) as "not evaluable." Forty-one (60%) patients did not have any prior conventional treatment. CONCLUSION: The challenge for submitters rests in their ability to supply sufficient documentation for the NCI BCS Program. The NCI BCS Program represents a unique avenue for the rigorous evaluation of "best cases" to identify complementary and alternative medicine modalities that are promising for prospective preclinical evaluation or prospective research.