Modalities to Deliver Cell Therapy for Treatment of Chronic Limb Threatening Ischemia.

Significance: Chronic limb threatening ischemia (CLTI) is a severe form of peripheral arterial disease (PAD) that is associated with high rates of morbidity and mortality, and especially limb loss. In patients with no options for revascularization, stem cell therapy is a promising treatment option. Recent Advances: Cell therapy directly delivered to the affected ischemic limb has been shown to be a safe, effective, and feasible therapeutic alternative for patients with severe PAD. Multiple methods for cell delivery, including local, regional, and combination approaches, have been examined in both pre-clinical studies and clinical trials. This review focuses on delivery modalities used in clinical trials that deliver cell therapy to patients with severe PAD. Critical Issues: Patients with CLTI are at high risk for complications of the disease, such as amputations, leading to a poor quality of life. Many of these patients do not have viable options for revascularization using traditional interventional or surgical methods. Clinical trials have shown therapeutic benefit for cell therapy in these patients, but methods of cell treatment are not standardized, including the method of cell delivery to the ischemic limb. Future Directions: The ideal delivery approach for stem cell therapy in PAD patients remains unclear. Further studies are needed to determine the best modality of cell delivery to maximize clinical benefits.

Carbon dioxide angiography during peripheral vascular interventions is associated with decreased cardiac and renal complications in patients with chronic kidney disease.

OBJECTIVE: Patients with chronic kidney disease (CKD) who undergo peripheral vascular interventions (PVI) with iodinated contrast are at higher risk of post-contrast acute kidney injury (PC-AKI). Carbon dioxide (CO2) angiography can reduce iodinated contrast volume usage in this patient population, but its impact on PC-AKI has not been studied. We hypothesize that CO2 angiography is associated with a decrease in PC-AKI in patients with advanced CKD. METHODS: The Vascular Quality Initiative PVI dataset from 2010 to 2021 was reviewed. Only patients with advanced CKD (estimated glomular filtration rate <45 ml/min/1.73 m2) treated for peripheral arterial disease were included. Propensity matching and multivariate logistic regression based on demographics, comorbidities, CKD stage, and indications were used to compare the outcomes of patients treated with and without CO2. RESULTS: There were 20,706 PVIs performed in patients with advanced CKD, and only 22% utilized CO2 angiography. Compared with patients treated without CO2, patients who underwent CO2 angiography were younger and less likely to be women or White, and more likely to have poor renal function, diabetes, cardiac comorbidities, and present with tissue loss. Propensity matching yielded well-matched groups with 4472 patients in each group. The procedural details after matching demonstrated 50% reduction in the volume of contrast used (32±33 vs 65±48 mL; P < .01). PVI with CO2 angiography was associated with lower rates of PC-AKI (3.9% vs 4.8%; P = .03) and cardiac complications (2.1% vs 2.9%; P = .03) without a significant difference in technical failure or major/minor amputations. Low contrast volumes (≤50 mL for CKD3, ≤20 mL for CKD4, and ≤9 mL for CKD5) are associated with reduced risk of PC-AKI (hazard ratio, 0.59; P < .01). CONCLUSIONS: CO2 angiography reduces iodinated contrast volume usage during PVI and is associated with decreased cardiac complications and PC-AKI. CO2 angiography is underutilized and should be considered for patients with advanced CKD who require endovascular therapy.

Risk Factors and Management of Hemodialysis Associated Distal Ischemia.

BACKGROUND: Hemodialysis-associated distal ischemia (HADI) is an uncommon, but significant complication after hemodialysis access creation that might require additional intervention. This study examines the risk factors for HADI and compares the outcomes of the different treatment modalities. METHODS: The Vascular Quality Initiative hemodialysis access (2011-2019) registry was reviewed. Patients were classified based on the occurrence of HADI requiring intervention or not, and their respective characteristics were compared. Multivariable logistic regression was used to identify independent factors associated with HADI. Kaplan Meier curves of secondary patency after different modalities of surgical revision were compared. RESULTS: There were 35,236 vascular access creations and 970 (2.75%) were complicated by HADI requiring intervention. Treatment was performed with access ligation in 224 patients (23%) and catheter-based techniques in 394 (41%). Open surgical revision consisted of banding in 127 (13%), distal revascularization interval ligation (DRIL) in 196 (20%), proximalization of arterial inflow (PAI) in 15 (1.5%), and revision using distal inflow (RUDI) in 14 (1.4%). Median time to HADI was 49 days (IQR 17 -91 days). Multivariate regression demonstrated that white race, female sex, peripheral artery disease, coronary artery disease, diabetes, post-procedure antiplatelets, prosthetic grafts, upper arm access, and target vein diameter greater than 4 mm were significantly associated with increased risk for HADI. When compared to procedures without HADI, access patency was decreased when revision (excluding access ligation) was performed (secondary patency at 12 months, HADI revision versus none: 89.0% vs. 92.4%, P <0.01). However, after multivariate Cox adjustment, revision for HADI was not independently significantly associated with access failure. CONCLUSION: HADI complicates 2.75% of hemodialysis access cases and is more likely in white females with diabetes and arterial disease after upper arm prosthetic graft placement. The patency of dialysis access does not seem to be negatively impacted by the various methods of surgical revision for HADI.

Arteriovenous Fistula-induced Cardiac Remodeling Shows Cardioprotective Features in Mice.

OBJECTIVE---: Arteriovenous fistulae (AVF) placed for hemodialysis have high flow rates that can stimulate left ventricular (LV) hypertrophy. LV hypertrophy generally portends poor cardiac outcomes, yet clinical studies point to superior cardiac-specific outcomes for patients with AVF when compared to other dialysis modalities. We hypothesize that AVF induce physiologic cardiac hypertrophy with cardioprotective features. METHODS---: 9-11 week C57Bl/6 male and female mice were treated with sham laparotomy or an aortocaval fistula via a 25Ga needle. Cardiac chamber size and function were assessed with serial echocardiography, and cardiac CTA. Hearts were harvested at 5 weeks post-operatively, and collagen content assessed with Masson's trichrome. Bulk mRNA sequencing was performed from LV of sham and AVF mice at 10 days. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (Qiagen) to identify affected pathways and predict downstream biological effects. RESULTS---: Mice with AVF had similar body weight and wet lung mass, but increased cardiac mass compared to sham-operated mice. AVF increased cardiac output while preserving LV systolic and diastolic function, as well as indices of right heart function; all 4 cardiac chambers were enlarged, with slight decrement in relative LV wall thickness. Histology showed preserved collagen density within each of the 4 chambers without areas of fibrosis. RNA sequencing captured 19,384 genes, of which 857 were significantly differentially expressed, including transcripts from extracellular matrix-related genes, ion channels, metabolism, and cardiac fetal genes. Top upstream regulatory molecules predicted include activation of angiogenic (Vegf, Akt1), pro-cardiomyocyte survival (Hgf, Foxm1, Erbb2, Lin9, Areg), and inflammation-related (CSF2, Tgfb1, TNF, Ifng, Ccr2, IL6) genes, as well as the inactivation of cardiomyocyte antiproliferative factors (Cdkn1a, FoxO3, α-catenin). Predicted downstream effects include reduction to heart damage, and increased arrhythmia, angiogenesis, and cardiogenesis. There were no significant sex-dependent differences in the AVF-stimulated cardiac adaptation. CONCLUSIONS---: AVF stimulate adaptive cardiac hypertrophy in wild-type mice without heart failure or pathological fibrosis. Transcriptional correlates suggest AVF-induced cardiac remodeling has some cardioprotective, although also arrhythmogenic features.

Quantitative not qualitative histology differentiates aneurysmal from nondilated ascending aortas and reveals a net gain of medial components.

Medial degeneration is a common histopathological finding in aortopathy and is considered a mechanism for dilatation. We investigated if medial degeneration is specific for sporadic thoracic aortic aneurysms versus nondilated aortas. Specimens were graded by pathologists, blinded to the clinical diagnosis, according to consensus histopathological criteria. The extent of medial degeneration by qualitative (semi-quantitative) assessment was not specific for aneurysmal compared to nondilated aortas. In contrast, blinded quantitative assessment of elastin amount and medial cell number distinguished aortic aneurysms and referent specimens, albeit with marked overlap in results. Specifically, the medial fraction of elastin decreased from dilution rather than loss of protein as cross-sectional amount was maintained while the cross-sectional number, though not density, of smooth muscle cells increased in proportion to expansion of the media. Furthermore, elastic lamellae did not thin and interlamellar distance did not diminish as expected for lumen dilatation, implying a net gain of lamellar elastin and intralamellar cells or extracellular matrix during aneurysmal wall remodeling. These findings support the concepts that: (1) medial degeneration need not induce aortic aneurysms, (2) adaptive responses to altered mechanical stresses increase medial tissue, and (3) greater turnover, not loss, of mural cells and extracellular matrix associates with aortic dilatation.

Strategies to Minimize Iodinated Contrast Volume in Patients with Chronic Kidney Disease Undergoing Peripheral Vascular Interventions.

Peripheral vascular interventions (PVI) utilize iodinated contrast medium (ICM) to visualize intravascular lesions and guide therapy. The use of ICM carries a risk of postcontrast acute kidney injury (PC-AKI), which is increased in the elderly and in patients with chronic kidney disease (CKD). Furthermore, the risk of PC-AKI increases with the volume of ICM used. This paper reports a 94-year-old patient with CKD stage 4 who presented with chronic limb threatening ischemia. He underwent successful endovascular revascularization using a combination of CO2 and dilute ICM (total volume = 6.5 mL). The case demonstrates strategies to minimize ICM during PVIs.

Activation of EphrinB2 Signaling Promotes Adaptive Venous Remodeling in Murine Arteriovenous Fistulae.

BACKGROUND: Arteriovenous fistulae (AVF) are the preferred mode of vascular access for hemodialysis. Before use, AVF remodel by thickening and dilating to achieve a functional conduit via an adaptive process characterized by expression of molecular markers characteristic of both venous and arterial identity. Although signaling via EphB4, a determinant of venous identity, mediates AVF maturation, the role of its counterpart EphrinB2, a determinant of arterial identity, remains unclear. We hypothesize that EphrinB2 signaling is active during AVF maturation and may be a mechanism of venous remodeling. METHODS: Aortocaval fistulae were created or sham laparotomy was performed in C57Bl/6 mice, and specimens were examined on Days 7 or 21. EphrinB2 reverse signaling was activated with EphB4-Fc applied periadventitially in vivo and in endothelial cell culture medium in vitro. Downstream signaling was assessed using immunoblotting and immunofluorescence. RESULTS: Venous remodeling during AVF maturation was characterized by increased expression of EphrinB2 as well as Akt1, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38. Activation of EphrinB2 with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and was associated with increased diameter and wall thickness in the AVF. Both mouse and human endothelial cells treated with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and increased endothelial cell tube formation and migration. CONCLUSIONS: Activation of EphrinB2 signaling by EphB4-Fc was associated with adaptive venous remodeling in vivo while activating endothelial cell function in vitro. Regulation of EphrinB2 signaling may be a new strategy to improve AVF maturation and patency.

Postcontrast Acute Kidney Injury after Peripheral Vascular Interventions in Kidney Transplant Recipients.

BACKGROUND: Postcontrast acute kidney injury (PC-AKI) is a feared complication of peripheral vascular interventions (PVIs), associated with increased mortality. Whether kidney transplant recipients (KTRs) are at increased risk of PC-AKI after PVI is unknown. This study analyzes the perioperative outcomes of KTR following PVI, with emphasis on the incidence and risk factors for PC-AKI. METHODS: The Vascular Quality Initiative files for PVI (2010-2018) were reviewed. Patients on dialysis were excluded. PC-AKI was defined by Vascular Quality Initiative as creatinine increase ≥0.5 mg/dL or new dialysis requirement. Characteristics of KTR and patients without kidney transplant were compared, and propensity score matching used to control for differences in baseline features. Multivariable logistic regression was used to define risk factors for PC-AKI, and survival was compared using Kaplan-Meier analysis. RESULTS: A total of 58,014 procedures were analyzed, including 641 (1%) procedures for KTR. The incidence of PC-AKI in KTR was 2.8% compared with 0.9% in patients without kidney transplants. Baseline warfarin use (odds ratio [OR] = 4.7) and poor allograft function (OR = 4.0) were significantly associated with increased risk for PC-AKI in KTR. Compared with a matched group of patients without kidney transplant, KTR had similar risk of PC-AKI and were more likely to develop postop myocardial infarction (OR = 4.3) but had lower in-hospital mortality (OR = 0.22). CONCLUSIONS: The incidence of PC-AKI in KTR is higher than the overall population undergoing PVI but is not elevated compared with propensity-matched patients without kidney transplant. PVI for peripheral artery disease in KTR is safe and associated with acceptable perioperative and long-term survival.

TGFß (Transforming Growth Factor-Beta)-Activated Kinase 1 Regulates Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) are the optimal conduit for hemodialysis access but have high rates of primary maturation failure. Successful AVF maturation requires wall thickening with deposition of ECM (extracellular matrix) including collagen and fibronectin, as well as lumen dilation. TAK1 (TGFß [transforming growth factor-beta]-activated kinase 1) is a mediator of noncanonical TGFß signaling and plays crucial roles in regulation of ECM production and deposition; therefore, we hypothesized that TAK1 regulates wall thickening and lumen dilation during AVF maturation. Approach and Results: In both human and mouse AVF, immunoreactivity of TAK1, JNK (c-Jun N-terminal kinase), p38, collagen 1, and fibronectin was significantly increased compared with control veins. Manipulation of TAK1 in vivo altered AVF wall thickening and luminal diameter; reduced TAK1 function was associated with reduced thickness and smaller diameter, whereas activation of TAK1 function was associated with increased thickness and larger diameter. Arterial magnitudes of laminar shear stress (20 dyne/cm2) activated noncanonical TGFß signaling including TAK1 phosphorylation in mouse endothelial cells. CONCLUSIONS: TAK1 is increased in AVF, and TAK1 manipulation in a mouse AVF model regulates AVF thickness and diameter. Targeting noncanonical TGFß signaling such as TAK1 might be a novel therapeutic approach to improve AVF maturation.

Co-culture of human fibroblasts, smooth muscle and endothelial cells promotes osteopontin induction in hypoxia.

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.

Venous Mechanical Properties After Arteriovenous Fistulae in Mice.

BACKGROUND: An arteriovenous fistula (AVF) exposes the outflow vein to arterial magnitudes and frequencies of blood pressure and flow, triggering molecular pathways that result in venous remodeling and AVF maturation. It is unknown, however, how venous remodeling, that is lumen dilation and wall thickening, affects venous mechanical properties. We hypothesized that a fistula is more compliant compared with a vein because of altered contributions of collagen and elastin to the mechanical properties. METHODS: Ephb4+/- and littermate wild-type (WT) male mice were treated with sham surgery or needle puncture to create an abdominal aortocaval fistulae. The thoracic inferior vena cava was harvested 3 wk postoperatively for mechanical testing and histological analyses of collagen and elastin. RESULTS: Mechanical testing of the thoracic inferior vena cava from Ephb4+/- and WT mice showed increased distensibility and increased compliance of downstream veins after AVF compared with sham. Although Ephb4+/- veins were thicker than WT veins at the baseline, after AVF, both Ephb4+/- and WT veins showed similar wall thickness as well as similar collagen and elastin area fractions, but increased collagen undulation compared with sham. CONCLUSIONS: Fistula-induced remodeling of the outflow vein results in circumferentially increased distensibility and compliance, likely due to post-translational modifications to collagen.

A mouse model of stenosis distal to an arteriovenous fistula recapitulates human central venous stenosis.

OBJECTIVE: Central venous stenosis (CVS) is a major cause of arteriovenous fistula (AVF) failure. However, central veins are relatively inaccessible to study with conventional Doppler ultrasound methods. To understand mechanisms underlying AVF failure owing to CVS, an animal model was established that creates a stenosis distal to an AVF. We hypothesized that this mouse model will show comparable morphology and physiology to human CVS. METHODS: An aortocaval fistula was created between the distal aorta and inferior vena cava (IVC); a stenosis was then created distal to the fistula by partial IVC ligation. Sham-operated animals, AVF without venous stenosis, and venous stenosis without AVF were used as controls. Physiologic properties of the IVC, both upstream and downstream of the stenosis, or the corresponding sites in models without stenosis, were assessed with ultrasound examination on days 0 to 21. The spectral broadening index was measured to assess the degree of disturbed shear stress. The IVC was harvested at day 21 and specimens were analyzed with immunofluorescence. RESULTS: The IVC diameter of mice with an AVF and stenosis showed increased upstream (P = .013), but decreased downstream diameter (P = .001) compared with mice with an AVF but without a stenosis, at all postoperative times (days 3-21). IVC wall thickness increased in mice with an AVF, compared with IVC without an AVF (upstream of stenosis: 13.9 µm vs 11.0 µm vs 4.5 µm vs 3.9 µm; P = .020; downstream of stenosis: 6.0 µm vs 6.6 µm vs µm 4.5 µm vs 3.8 µm; P = .002; AVF with stenosis, AVF, stenosis, sham, respectively). AVF patency significantly decreased in mice with an AVF and stenosis by day 21 (50% vs 90%; P = .048). The IVC of mice with AVF and stenosis showed a venous waveform with pulsatility as well as enhanced velocity at and downstream of the stenosis; similar waveforms were observed in a human case of CVS. Downstream to the stenosis, the spectral broadening index was significantly higher compared with mice with AVF alone (1.06 vs 0.78; P = .011; day 21), and there was a trend towards less immunoreactivity of both Krüppel-like factor 2 and phosphorylated-endothelial nitric oxide synthase compared with mice with an AVF alone. CONCLUSIONS: Partial IVC ligation distal to a mouse aortocaval fistula alters the fistula diameter and wall thickness, decreases patency, and increases distal disturbed flow compared with fistulae without a distal stenosis. Our mouse model of stenosis distal to an AVF may be a faithful representation of human CVS that shows similar morphology and physiology, including disturbed shear stress.

Risk factors and safe contrast volume thresholds for postcontrast acute kidney injury after peripheral vascular interventions.

OBJECTIVE: Postcontrast acute kidney injury (PC-AKI) is a dreaded complication of peripheral vascular interventions (PVIs) that depends on the volume of contrast administered as well as a patient's baseline kidney function. However, there is currently no guidance on the volume of contrast that is considered safe especially for patients with advanced chronic kidney disease (CKD). This study aims to characterize the incidence, risk factors for, and outcomes after PC-AKI and define thresholds of safety for contrast volume. METHODS: The Vascular Quality Initiative files for PVI (2010-2018) were reviewed. Patients on dialysis, with renal transplants, or who developed a bleeding complication were excluded. Only records with complete data on baseline creatinine, contrast volume, and PC-AKI (creatinine increase of ≥0.5 mg/dL, or new dialysis requirement) were included. The cumulative incidence of PC-AKI with contrast volume at each stage of CKD was derived. A safe threshold for contrast volume was defined as the volume at which the cumulative incidence of PC-AKI is 0.5% or less. Multivariable logistic regression was used to define risk factors for PC-AKI, and survival analysis was performed using Kaplan-Meier and multivariable Cox proportional hazards regression. RESULTS: A total of 53,780 procedures were included. There were 16,062 patients (29%) with normal kidney function or CKD1, 21,769 (39%) with CKD2, 14,234 (25%) with CKD3, 1471 (3%) with CKD4, and 199 (<1%) with CKD5. The incidence of PC-AKI was 0.9% and increased with each stage of CKD (CKD1, 0.39%; CKD2, 0.45%; CKD3, 1.5%; CKD4, 4.3%; and CKD5, 7.5%). The safe thresholds for contrast volume for advanced CKD were 50, 20, and 9 mL for CKD3, CKD4, and CKD5, respectively. Regression analysis demonstrated that white race (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.54-0.82) and elective surgery (OR, 0.77; 95% CI, 0.62-0.95) were associated with decreased risk of PC-AKI, whereas inpatient status (OR, 14.5; 95% CI, 9.97-21.2), diabetes (OR, 1.27; 95% CI, 1.02-1.58), advanced CKD (CKD3: OR, 3.65; 95% CI, 2.68-4.98; CKD4: OR, 6.98; 95% CI, 4.72-10.3; CKD5: OR, 8.94; 95% CI, 4.53-17.6), critical limb ischemia (OR, 1.51; 95% CI, 1.14-2.00), acute limb ischemia (OR, 2.47; 95% CI, 1.70-3.59), and contrast-to-eGFR ratio (CGR) (2 ≤ CGR < 3: OR, 1.33; 95% CI, 1.02-1.74; 3 ≤ CGR < 4: OR, 1.90; 95% CI, 1.32-2.75; CGR ≥ 4: OR, 1.79; 95% CI, 1.18-2.70) were significantly associated with increased risk for PC-AKI. Patients who developed PC-AKI had worse in-hospital (16.1% vs 0.45%; P < .01) mortality and long-term survival (log-rank P < .01) compared with those without PC-AKI. CONCLUSIONS: PVI are associated with low risk of PC-AKI that significantly increases when patients with advanced CKD undergo high acuity cases. Given the strong association with short-term and long-term mortality, risk of PC-AKI should be minimized by using safe thresholds of contrast volume.

Renal Failure After Cardiac Operations: Not All Acute Kidney Injury Is the Same.

BACKGROUND: The Society of Thoracic Surgeons (STS) database does not distinguish between a decline in creatinine clearance vs new hemodialysis (HD) when qualifying acute renal failure (ARF) after a cardiac operation. We hypothesized that patients requiring HD experience significantly greater postoperative morbidity and death. METHODS: We included all patients who underwent STS index cardiac operations at our institution from 2008 to March 2015 and did not have preexisting renal failure (creatinine >4.0 mg/dL or preoperative HD). We identified patients meeting STS criteria for ARF: threefold rise in serum creatinine, creatinine exceeding 4.0 mg/dL (non-HD ARF) with minimum rise of 0.5 mg/dL, or HD (ARF-HD). After propensity matching non-HD ARF and ARF-HD groups across 14 variables (including baseline glomerular filtration rate), we compared incidences of our primary outcome, death, and secondary outcomes, intensive care unit (ICU) and hospital length of stay (LOS), and discharge to a location other than home. RESULTS: Among 4,154 study patients, we identified 113 (2.7%) that experienced new-onset non-HD ARF (n = 57) or ARF-HD (n = 56) postoperatively. Propensity matching resulted in 51 well-matched pairs who experienced non-HD ARF or ARF-HD (all p > 0.10). Patients requiring HD suffered significantly greater operative mortality (67% vs 22%, p < 0.01), longer ICU LOS (326 vs 176 hours, p < 0.01), and greater postoperative hospital LOS (34 vs 17 days, p < 0.01). ARF-HD patients also demonstrated a trend toward higher rates of discharge to a location other than home (71% vs 45%, p = 0.08). CONCLUSIONS: After cardiac operations, patients who experienced ARF-HD experienced triple the mortality and double the ICU and postoperative hospital LOS compared with patients who experienced non-HD ARF.

Long-Term Outcomes of 98 Surgically Resected Metastatic Tumors in the Pancreas.

PURPOSE: The goal of this study was to assess the outcomes and characteristics of patients who underwent pancreatectomy for metastatic disease to the pancreas. METHODS: Patients who underwent surgical resection of metastatic disease to the pancreas from 1988 to 2016 were identified using a prospectively maintained database. Data on clinicopathological features and outcomes of these patients were analyzed. Cox proportional hazard models were employed to identify patient-specific risk factors that influence survival. RESULTS: Ninety-seven patients underwent 98 pancreatic metastasectomies from July 1988 through March 2016 for metastatic disease from 13 different primary cancers. Pancreaticoduodenectomy, distal pancreatectomy, and total pancreatectomy were performed in 49 (50 %), 37 (38 %), and 12 (12 %) patients, respectively. Postoperative complications occurred in 55 (56 %) patients, while 3 (3 %) perioperative deaths occurred. Median follow-up was 2.0 years, with a median survival of 3.2 years. Multivariate analysis revealed that older patients [hazard ratio (HR) 1.04/year; p = 0.006], non-renal cell carcinomas (HR 5.07; p < 0.001), vascular invasion (HR 3.53; p < 0.001), and positive resection margins (HR 2.62; p = 0.008) were independently associated with an increased risk of mortality. CONCLUSIONS: Pancreatic metastasectomy is safe and feasible in well-selected patients and is associated with acceptable long-term survival.

Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

Large Ca²âº-dependent facilitation of Ca(V)2.1 channels revealed by Ca²âº photo-uncaging.

KEY POINTS: CaV 2.1 channels constitute a dominant Ca(2+) entry pathway into brain neurons, triggering downstream Ca(2+) -dependent processes such as neurotransmitter release. CaV 2.1 is itself modulated by Ca(2+) , resulting in activity-dependent enhancement of channel opening termed Ca(2+) -dependent facilitation (CDF). Real-time Ca(2+) imaging and Ca(2+) uncaging here reveal that CDF turns out to be strikingly faster, more Ca(2+) sensitive, and larger than anticipated on previous grounds. Robust resolution of the quantitative profile of CDF enables deduction of a realistic biophysical model for this process. These results suggest that CaV 2.1 CDF would figure most prominently in short-term synaptic plasticity and cerebellar Purkinje cell rhythmicity. ABSTRACT: CaV 2.1 (P-type) voltage-gated Ca(2+) channels constitute a major source of neuronal Ca(2+) current, strongly influencing rhythmicity and triggering neurotransmitter release throughout the central nervous system. Fitting with such stature among Ca(2+) entry pathways, CaV 2.1 is itself feedback regulated by intracellular Ca(2+) , acting through calmodulin to facilitate channel opening. The precise neurophysiological role of this calcium-dependent facilitation (CDF) remains uncertain, however, in large measure because the very magnitude, Ca(2+) dependence and kinetics of CDF have resisted quantification by conventional means. Here, we utilize the photo-uncaging of Ca(2+) with CaV 2.1 channels fluxing Li(+) currents, so that voltage-dependent activation of channel gating is no longer conflated with Ca(2+) entry, and CDF is then driven solely by light-induced increases in Ca(2+) . By using this strategy, we now find that CDF can be unexpectedly large, enhancing currents by as much as twofold at physiological voltages. CDF is steeply Ca(2+) dependent, with a Hill coefficient of approximately two, a half-maximal effect reached by nearly 500 nm Ca(2+) , and Ca(2+) on/off kinetics in the order of milliseconds to tens of milliseconds. These properties were established for both native P-type currents in cerebellar Purkinje neurons, as well as their recombinant channel counterparts under heterologous expression. Such features suggest that CDF of CaV 2.1 channels may substantially enhance the regularity of rhythmic firing in cerebellar Purkinje neurons, where regularity is believed crucial for motor coordination. In addition, this degree of extensive CDF would be poised to exert large order-of-magnitude effects on short-term synaptic plasticity via rapid modulation of presynaptic Ca(2+) entry.

Optical mapping of optogenetically shaped cardiac action potentials.

Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution. Here, we make progress by combining optical mapping of action potentials with concurrent activation of channelrhodopsin-2 (ChR2) or halorhodopsin (eNpHR3.0), via an all-optical system applied to monolayers of neonatal rat ventricular myocytes (NRVM). Additionally, we explore the capability of ChR2 and eNpHR3.0 to shape action-potential waveforms, potentially aiding the study of short/long QT syndromes that result from abnormal changes in action potential duration (APD). These results show the promise of an all-optical system to acquire action potentials with precise temporal optogenetics control, achieving a long-sought flexibility beyond the means of conventional electrical stimulation.