The susceptibility of the aortic root: porcine aortic rupture testing under cardiopulmonary bypass

Background In our earlier study on the functional limits of the aneurysmal aortic root we determined the pig root is susceptible to failure at high aortic pressures levels. We established a pig rupture model using cardiopulmonary bypass to determine the most susceptible region of the aortic root under the highest pressures achievable using continuous flow, and what changes occur in these regions on a macroscopic and histological level. This information may help guide clinical management of aortic root and ascending aorta pathology. Methods Five pigs underwent 4D flow MRI imaging pre surgery to determine vasopressor induced wall sheer stress and flow parameters. All pigs were then placed on cardiopulmonary bypass (CPB) via median sternotomy, and maximal aortic root and ascending aorta flows were initiated until rupture or failure, to determine the most susceptible region of the aorta. The heart was explanted and analysed histologically to determine if histological changes mirror the macroscopic observations. Results The magnetic resonance imaging (MRI) aortic flow and wall sheer stress (WSS) increased significantly in all regions of the aorta, and the median maximal pressures obtained during cardiopulmonary bypass was 497 mmHg and median maximal flows was 3.96 L/m. The area of failure in all experiments was the non-coronary cusp of the aortic valve. Collagen and elastin composition (%) was greatest in the proximal regions of the aorta. Collagen I and III showed greatest content in the inner aortic root and ascending aorta regions. Conclusions This unique porcine model shows that the aortic root is most susceptible to failure at high continuous aortic pressures, supported histologically by different changes in collagen content and subtypes in the aortic root. With further analysis, this information could guide management of the aortic root in disease.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

The susceptibility of the aortic root: porcine aortic rupture testing under cardiopulmonary bypass

Background In our earlier study on the functional limits of the aneurysmal aortic root we determined the pig root is susceptible to failure at high aortic pressures levels. We established a pig rupture model using cardiopulmonary bypass to determine the most susceptible region of the aortic root under the highest pressures achievable using continuous flow, and what changes occur in these regions on a macroscopic and histological level. This information may help guide clinical management of aortic root and ascending aorta pathology. Methods Five pigs underwent 4D flow MRI imaging pre surgery to determine vasopressor induced wall sheer stress and flow parameters. All pigs were then placed on cardiopulmonary bypass (CPB) via median sternotomy, and maximal aortic root and ascending aorta flows were initiated until rupture or failure, to determine the most susceptible region of the aorta. The heart was explanted and analysed histologically to determine if histological changes mirror the macroscopic observations. Results The magnetic resonance imaging (MRI) aortic flow and wall sheer stress (WSS) increased significantly in all regions of the aorta, and the median maximal pressures obtained during cardiopulmonary bypass was 497mmHg and median maximal flows was 3.96L/m. The area of failure in all experiments was the non-coronary cusp of the aortic valve. Collagen and elastin composition (%) was greatest in the proximal regions of the aorta. Collagen I and III showed greatest content in the inner aortic root and ascending aorta regions. Conclusions This unique porcine model shows that the aortic root is most susceptible to failure at high continuous aortic pressures, supported histologically by different changes in collagen content and subtypes in the aortic root. With further analysis, this information could guide management of the aortic root in disease.

Abstract 16504: Low Disturbed Flow Induces Dynamic Immune Compartment Alterations in Atherogenesis as Revealed by Single Cell Rna-seq

Low disturbed blood flow (LDF) is a critical contributing factor to atherogenesis but its direct impact on the immune compartment was not well-depict. To fill this knowledge gap, we adopted scRNA-seq to capture sheer-stress induced immune responses during atherogenesis. A partial carotid artery ligation (PCAL) model was selected for its paired comparison of carotid arteries with normal flow (NF) or LDF. Indeed, we observed drastic changes in both endothelial and immune compartment. Macrophages were the most significantly increased population induced by LDF (from 4% to 12% of CD45+ cells) with two well-separated subsets (Mac-c8, Mac-c9). MacSpectrum analyses revealed that Mac-c8 displayed higher inflammatory states than the lipid-laden Mac-c9. Interestingly, three T subtypes displayed unique flow-induced enrichment patterns that were selectively enriched in LDF but not in the NF condition. Furthermore, we created an original algorithms to evaluate the impact of sheer-stress on membrane protein-mediated cell-cell interaction among all cell types in the atheroma. Several pairs of molecular interactions were identified, including multiple APP-ligands interaction pairs and those in BAG2 Signaling. Moreover, signature genes identified in these LDF-induced T cells displayed high correlation to the plaque severity in human artery-aorta samples. Collectively, our study provided a high-resolution and focused analyses of sheer-stress induced immune cell action during atherogenesis. This is also the first identification of unique T subsets, to our knowledge, that are enriched in arterial wall exposed to low and disturbed flow. Further characterization of these cells will provide valuable information to understand and therapeutically treat atherogenesis.

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

ABSTRACTNeutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (MFN2) regulates neutrophil homeostasis and chemotaxis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue, trapped in the vasculature in zebrafish embryos, and not capable of chemotaxis. Consistent with this, human neutrophil-like cells that are deficient for MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis on 2D surfaces. Deletion of MFN2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mechanistically, MFN2-deficient neutrophil-like cells display disrupted mitochondria–ER interaction, heightened intracellular Ca2+ levels and elevated Rac activation after chemokine stimulation. Restoring a mitochondria–ER tether rescues the abnormal Ca2+ levels, Rac hyperactivation and chemotaxis defect resulting from MFN2 depletion. Finally, inhibition of Rac activation restores chemotaxis in MFN2-deficient neutrophils. Taken together, we have identified that MFN2 regulates neutrophil migration via maintaining the mitochondria–ER interaction to suppress Rac activation, and uncovered a previously unrecognized role of MFN2 in regulating cell migration and the actin cytoskeleton.This article has an associated First Person interview with the first authors of the paper.

Cellular Mechanisms of Metabolic Remodeling During Fluid Sheer Stress-Induced Metastasis

Objectives Fluid sheer stress (FSS) is a physical stimuli of circulating tumor cells responsible for development of and progression to cancer. FSS is reported to enhance chemoresistance and proliferation in breast cancer cells. However, cellular mechanisms explaining how FSS contributes to the metastatic phenotype of breast cancer cell are less known. Chemoresistance is highly dependent upon active transport systems, and cell division and growth require ATP. In this study, we hypothesize that FSS contributes to mitochondrial remodeling and leads to alterations in energy metabolism which favor metastasis. Methods MDA-MB-231 human breast cancer cells were exposed to fluid sheer stress (FSS). MDA-MB-231 cells were then grown in culture media for 24 h, and intracellular energy (ATP) and abundance of ATP synthase were analyzed. Results FSS significantly increases intracellular ATP in MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP indicating remodeling and decreased reliance on mitochondrial energy metabolism. We then quantified the abundance of ATP synthase, the key enzyme complex that produces mitochondrial ATP. FSS significantly decreased protein levels of the c-subunit of ATP synthase. Conclusions Our data show that FSS causes metabolic remodeling of mitochondria-dependent ATP production. We suggest that the c-subunit of ATP synthase is an important target of FSS-mediated metastasis. Strategies to enhance the abundance or activity of the c-subunit may prevent metabolic remodeling-associated with metastasis in FSS-exposed circulating cancer cells. Funding Sources Alabama Life Research Institute (ALRI) 14,565.

Modeling of the minimum cutting thickness in micro cutting with consideration of the friction around the cutting zone

Friction modeling between the tool and the workpiece plays an important role in predicting the minimum cutting thickness during TC4 micro machining and finite element method (FEM) cutting simulation. In this study, a new three-region friction modeling is proposed to illustrate the material flow mechanism around the friction zone in micro cutting; estimate the stress distributions on the rake, edge, and clearance faces of the tool; and predict the stagnation point location and the minimum cutting thickness. The friction modeling is established by determining the distribution of normal and shear stress. Then, it is applied to calculate the stagnation point location on the edge face and predict the minimum cutting thickness. The stagnation point and the minimum cutting thickness are also observed and illustrated in the FEM simulation. Micro cutting experiments are conducted to validate the accuracy of the friction and the minimum cutting thickness modeling. Comparison results show that the proposed friction model illustrates the relationship between the normal and sheer stress on the tool surface, thereby validating the modeling method of the minimum cutting thickness in micro cutting.

Double Whammy: Pigment Nephropathy and Warfarin-related Nephropathy As Aetiology for Acute Kidney Injury in a Patient With Mechanical Heart Valves

It is well known that patients with mechanical heart valves may develop sheer stress related hemolysis and consequent pigment related nephropathy. Warfarin Related Nephropathy (WRN) is a relatively new entity and defined as Acute Kidney Injury (AKI) in the setting of an INR of > 3.0 excluding other obvious etiologies. A biopsy diagnosis of WRN is conducted when red blood cell casts are noted filling and blocking the tubules; additionally, glomerular hemorrhage may be observed. We describe a patient with mechanical heart valves on oral anticoagulation who developed both pigment nephropathy and WRN causing AKI.