Blood Flow Shear Research Articles

Abstract 4136901: Mechanosensitive channel protein Piezo1 synergizes with TLR4 to enhance osteogenic responses in human aortic valve interstitial cells

Background: Calcific aortic valve disease (CAVD), a common heart valve disease in the elderly, has a complex pathogenesis. Abnormal blood flow shear can be regarded as an important factor in the development of CAVD, and a key molecule that responds to mechanical stress is the mechanosensitive ion channel protein Piezo1; however, the role of Piezo1 in CAVD has not been clarified. Methods and results: We collected CAVD patients and their age- and sex-matched controls, and compared the differences in the protein expression levels of Piezo1 and TLR4 between the two using Western blot and other techniques, and found that the expression of Piezo1 and TLR4 proteins was elevated in the CAVD group. We gave LPS or Piezo1 agonist Yoda1 to stimulate aortic valve mesenchymal stromal cells, respectively, and the Western blot results showed that ALP and Runx2 expression were elevated after LPS and Yoda1 stimulation, and immunofluorescence and alizarin red staining showed similar results. Inhibition of TLR4 or Piezo1 attenuated the osteogenic differentiation of AVICs. Cellular immunofluorescence results showed that TLR4 could be transported from the Golgi to the cell membrane under Yoda stimulation, and Golgi-cell membrane transport of TLR4 was attenuated after Rab3b knockdown by siRNA. After first pre-stimulation with Yoda1 and then stimulation with LPS, Western blot and immunofluorescence results showed that the protein levels of ALP and Runx2 as well as NF-κB phosphorylation were to be significantly increased. Conclusion: Stimulation of Piezo1 and TLR4 can enhance the osteogenic response of AVICs. Stimulation of Piezo1 enhances the transport of TLR4 from the Golgi to the cell membrane via Rab3b, and Piezo1 synergistically enhances the osteogenic response of AVICs with TLR4, thus promoting the development of CAVD.

Influence of disturbed blood flow on endothelial function in people living with HIV

Abstract Introduction People infected with human immunodeficiency virus (HIV) are more susceptible to endothelial dysfunction due to the virus itself, side effects of the antiretroviral therapy and classical risk factors. Endothelial damage is most prominent at the locations where laminar blood flow is disturbed and oscillatory shear stress with retrograde blood flow is dominating. Purpose The goal of this study was to assess the endothelial function in people with HIV, and to determine whether their endothelium is more susceptible to a short-term increase in retrograde blood flow in comparison to healthy individuals. Methods The study included 62 people with HIV infection and 38 people without HIV infection, in whom endothelial function was assessed using flow-mediated dilatation, which was presented in absolute (FMD) and relative (FMD%) values. The intervention produced oscillatory shear stress and retrograde blood flow during 20 minutes, after which the endothelial function assessment was repeated. Results Baseline FMD (0.30±0.07 vs. 0.36±0.07 mm; p<0.05) and FMD% (6.84±1.57 vs. 7.89±1.19 %, p<0.05) were lower in the HIV group in compare to controls. FMD and FMD% decreased in the HIV group by 0.03 mm and 0.7% (p<0.05), while in the control group they decreased by 0.09 mm and 2.0% (p<0.05). The post-intervention FMD and FMD% were not different between the groups (p>0.05). Conclusion People with HIV infection express worse endothelial function in compare to healthy individuals. Retrograde blood flow and oscillatory shear stress deteriorate endothelial function in both HIV patients and healthy controls, although this decline is more pronounced in healthy individuals.

Biomechanics cerebral and carotid arteries: aneurysm development and atherosclerotic plaque detachment in the presence of combined pathologies

Today, the main cause of mortality in the world remains cardiovascular diseases. In this case, the combined pathology of the neck and head arteries, in which atherosclerosis affects the carotid arteries and there is a change in blood flow in the vertebral arteries or in the circle of Willis due to aplasia of the communicating artery, significantly increases the risk of aneurysms formation and their subsequent rupture. Assessing the risk of complications in this type of pathological condition may allow the physician to initiate timely preventive measures in specific clinical cases. The paper presents the results of modelling various variants of combined pathologies in the arteries of the neck and head. The segment under consideration includes sections of the common carotid, external and internal carotid arteries, as well as vessels of the circle of Willis and the basilar artery. The main idea of the study was to create a prognostic model to assess the risks of aneurysm formation in the circle of Willis and atherosclerotic plaque rupture in the carotid arteries. Such a system may be useful for physicians in routine practice when planning treatment tactics in patients with combined pathologies of the arterial channel. Based on the literature analysis, a number of pathologies of the cerebral and carotid arteries, most frequently occurring in various combinations, were identified: stenoses of the ICA (30 % and 70 %) and aplasia (absence) of one of the connecting arteries were considered from the point of view of geometrical features. In addition, the character of blood flow in the basilar artery, which can change its direction in steal-syndrome, was taken into account. Based on the analysis of blood flow and wall shear stress, two degrees of risk of aneurysm formation were distinguished. High risk corresponds to cases for which both of these values were statistically significantly different from the norm. Medium risk corresponds to cases for which only one of the values was statistically significantly different from the norm. For models containing atherosclerotic plaques, data on shear and normal stresses on the surfaces of atherosclerotic plaques were tabulated. The risk of plaque rupture on each side was determined for each case by analysing the result sets 11 combinations of pathological conditions with increased risk were indicated.

Assessment of perianal fistulizing Crohn's disease activity with endoanal ultrasound: A retrospective cohort study.

Perianal fistulas pose dual challenges to Crohn's disease (CD) patients. Low patient compliance due to the complexity of existing examination methods plagues the treatment and follow-up management of perianal CD. To determine the accuracy of endoanal ultrasound (EUS) and shear wave elastography (SWE) for evaluating perianal fistulizing CD (PFCD) activity. This was a retrospective cohort study. A total of 67 patients from August 2022 to December 2023 diagnosed with CD were divided into three groups: Non-anal fistula group (n = 23), low-activity perianal fistulas [n = 19, perianal disease activity index (PDAI) ≤ 4], high-activity perianal fistulas (n = 25, PDAI > 4) based on the PDAI. All patients underwent assessments including EUS + SWE, pelvic magnetic resonance [pelvic magnetic resonance imaging (MRI)], C-reactive protein, fecal calprotectin, CD activity index, PDAI. The percentage of fistulas indicated by pelvic MRI and EUS was consistent at 82%, and there was good consistency in the classification of perianal fistulas (Kappa = 0.752, P < 0.001). Significant differences were observed in the blood flow Limberg score (χ 2 = 8.903, P < 0.05) and shear wave velocity (t = 2.467, P < 0.05) between group 2 and 3. Shear wave velocity showed a strong negative correlation with magnetic resonance novel index for fistula imaging in CD (Magnifi-CD) score (r = -0.676, P < 0.001), a weak negative correlation with the PDAI score (r = -0.386, P < 0.05), and a weak correlation between the Limberg score and the PDAI score (r = 0.368, P < 0.05). EUS combined with SWE offers a superior method for detecting and quantitating the activity of perianal fistulas in CD patients. It may be the ideal tool to assess PFCD activity objectively for management strategies.

"One-Stone-Three-Birds" H2S-Photothermal Therapy for Enhanced Thrombolysis and Vascular Healing.

Thrombus causes a serious condition characterized by the formation of blood clots in blood vessels or heart, potentially leading to life-threatening emergencies. Photothermal therapy (PTT) serves as a treatment for thrombosis that provides noninvasive thrombus dissolution and fewer bleeding side effects.However, the high temperatures generated by PTT can exacerbate vascular inflammation and promote thrombus recurrence. In this study, a photothermal hydrogen sulfide (H2S) nanogenerator (PSA@ADT-OH) is constructed using a perylene-cored photothermal agent (PSA) coassembled with a H2S donor ADT-OH. The system PSA@ADT-OH demonstrates outstanding targeting and accumulation efficiency against blood flow shear forces. It also provides sustained H2S release at thrombus sites, contributing to antiplatelet aggregation, reactive oxygen species clearance, and vascular healing. This approach opens up new possibilities for advanced thrombus treatment.

Flow shear force destabilizes carotid plaques by affecting CHOP and GRP78 proteins

BackgroundVarious factors, including blood, inflammatory, infectious, and immune factors, can cause ischemic stroke. However, the primary cause is often the instability of cervical arteriosclerosis plaque. It is estimated that 18-25% of ischemic strokes are caused by the rupture of carotid plaque.1 Plaque stability is crucial in determining patient prognosis. Developing a highly accurate, non-invasive, or minimally invasive technique to assess carotid plaque stability is crucial for diagnosing and treating stroke.Previous research by our group has demonstrated that the expression levels of CHOP (C/EBP homologous protein) and GRP78 (glucose-regulated protein 78) are correlated with the stability of atherosclerotic plaques.2 ObjectThis research assesses changes in GRP78 and CHOP expressions in human umbilical vein endothelial cells(HUVEC) following experiments within the hemodynamic influencing factors test system. Additionally, it includes conducting an empirical study on the impact of blood flow shear force on the stability of human carotid atherosclerotic plaques. The objective is to explore the implications of blood flow shear force on the stability of carotid atherosclerotic plaques. MethodThe hemodynamic influencing factors test bench system was configured with low (Group A, 4 dyns/cm²), medium (Group B, 8 dyns/cm²), and high shear force groups (Group C, 12 dyns/cm²). Relative expression levels of GRP78 and CHOP proteins in human umbilical vein endothelial cells were measured using Western blot analysis, and quantitative analysis of GRP78 and CHOP mRNA was conducted using RT-qPCR. Meanwhile, plaques from 60 carotid artery patients, retrieved via Carotid Endarterectomy (CEA), were classified into stable (S) and unstable (U) groups based on pathological criteria. Shear force at the carotid bifurcation was measured preoperatively using ultrasound. Western blot and RT-qPCR were used to analyze the relative expression levels of GRP78 and CHOP proteins and mRNA, respectively, in the plaque specimens from both groups. ResultExpression levels of GRP78, CHOP proteins, and their mRNAs were assessed in groups A, B, and C via Western blot and RT-qPCR. Results showed that in the low-shear group, all markers were elevated in group A compared to groups B and C. Statistical analysis revealed significantly lower shear forces at the carotid bifurcation in group U compared to group S. In group U plaques, GRP78 and CHOP expressions were significantly higher in group U than in group S. ConclusionBlood flow shear forces variably affect the expression of GRP78 and CHOP proteins, as well as their mRNA levels, in vascular endothelial cells. The lower the shear force and fluid flow rate, the higher the expression of GRP78 and CHOP, potentially leading to endoplasmic reticulum stress(ERS), which may destabilize the plaque.

Acute Effects of Skeletal Muscle Electrical Stimulation on Central and Lower Extremity Hemodynamics.

Belt electrode-skeletal muscle electrical stimulation (B-SES) is a treatment prescribed for individuals with difficulty performing exercise therapy that improves muscle strength, exercise tolerance, and glucose metabolism. However, the effects of B-SES on the hemodynamics of the central and lower extremity conduit arteries have not been studied. Therefore, this study compared the acute effects of B-SES on the central and lower extremity conduit arteries in healthy young males. This randomized crossover study included nine healthy young males (mean age: 21.0±1.1 years). Participants were assigned to the following experimental conditions, with a washout period of one week: condition 1 included 20 min of electrical stimulation of the lower extremity at the participant's sensation threshold intensity (Sham, n=9)and condition 2included 20 min of electrical stimulation of the lower extremity at the maximum intensity the participant can tolerate (B-SES, n=9). The heart rate (HR), stroke volume (SV), cardiac output (CO), mean arterial pressure (MAP), and total peripheral vascular resistance (TPR) were measured as central hemodynamics. The hemodynamics of the lower extremity conduit arteries were measured and calculated for the shallow femoral artery (SFA), including vessel diameter, mean blood flow velocity (MBFV), shear rate (SR), and mean blood flow (MBF) rate. These indices were measured before stimulation (Pre), 10 min after the start of stimulation (Stimulating), and immediately after the end of stimulation (Post). These indices were compared using a repeated two-way analysis of variance. In B-SES, HR (Pre: 63.2±8.6; Stimulating: 73.7±6.9; Post: 70.0±4.2 bpm, p<0.01), CO (Pre: 5.1±1.0; Stimulating: 6.5±1.5, p<0.01; Post: 6.3±1.2 L/min, p=0.02), and MAP (Pre: 104.0±11.5; Stimulating: 116.4±10.8, p<0.01; Post: 109.6±9.7 mmHg, p=0.02) increased significantly. In addition, B-SES significantly increased MBFV (Pre: 19.2±4.0; Stimulating: 50.5±14.9; Post: 30.1±4.0 cm/s, p<0.01), SR (Pre: 118.9±28.8; Stimulating: 302.7±91. 2, p<0.01; Post: 182.1±70.1/s, p=0.02), and MBF (Pre: 382.0±61.5; Stimulating: 1009.6±321.4; Post: 626.8±176.6 mL/min, p<0.01). However, there were no significant changes in SV and TPR. The findings of this study indicatethat B-SES in healthy young males increases CO without increasing SV or TPR and improves the MBFV and SR in the SFA.

Abstract 153: Multi-parametric Thrombus Profiling Microfluidics Uncovers Mechanobiology Of Hypertension And Aging-related Thrombosis

Arterial thrombosis, which represents a critical complication of cardiovascular diseases, is a leading cause of death and disability worldwide. A symbolic feature of arterial thrombosis is large-scale platelet aggregation due to high shear stress generated by stenosis. However, current hematological tests cannot evaluate arterial thrombosis in such physiological settings. To bridge this gap, we developed a microfluidics-based thrombus profiling assay (Fig. 1A) that combines multi-fluorescence imaging with a stenotic channel design to comprehensively characterize thrombi formed in high shear blood flow (Fig. 1B, C). Thrombi are characterized by a 7-dimensional profile indicating the thrombus size (Plt) and levels of fibrinogen (Fg), VWF, P-selectin, phosphatidylserine (PS) and extended (E + ) and activated α IIb β 3 in the thrombus (Fig. 1D). Using this assay, we discovered intensified thrombus formation in subjects with hypertension and/or aging (Fig. 1D). We also found extended integrin α IIb β 3 as a superior biomarker for platelet hyperreactivity than P-selectin and activated integrin α IIb β 3 and a potential predictor for arterial thrombosis (Fig. 1E). By studying the effects of anti-thrombotic agents NMC4 and 7E3 on hypertension patients’ blood, our work reveals a ‘treatment mismatch’ phenomenon that can seriously affect the efficacy and safety of anti-thrombotic drugs (Fig. 1F). Our findings provide mechanistic insights into the high risk of arterial thrombosis and antiplatelet resistance in populations with hypertension and aging. The discovered ‘treatment mismatch’ heralds a new era in thrombosis management where patients’ thrombus profiles dictate personalized therapeutic decisions to optimize treatment efficacy and reduce adverse outcomes. The outstanding performance of our assay underscores its translational potential for anti-thrombotic drug screening, thrombosis diagnosis, and personalized anti-thrombotic regimen selection.

Oscillatory shear stress promotes endothelial senescence and atherosclerosis via STING activation

Endothelial dysfunction is an initiating factor in atherosclerosis. Endothelial cells (ECs) are constantly subject to blood flow shear stress, and atherosclerotic plaques tend to occur in aortic bends or bifurcations impaired by low oscillatory shear stress (OSS). However, the mechanism that how OSS affects the initiation and progression of atherosclerosis remains to be explored. Here, we first reported that OSS can promote endothelial dysfunction and atherogenesis in vivo and in vitro by activating STING pathway. Mechanistically, at atherosclerosis-prone areas, OSS caused mitochondria damage in ECs, leading to the leakage of mitochondrial DNA (mtDNA) into the cytoplasm. The cytoplasmic mtDNA was recognized by cGAS to produce cGAMP, activating the STING pathway and leading to endothelial senescence, which resulted in endothelial dysfunction and atherosclerosis. We found that STING was activated in plaques of atherosclerotic patients and in aortic arch ECs of high-fat diet (HFD)-fed ApoeKO mice, as well as in ECs exposed to OSS. STING-specific deficiency in ECs attenuates endothelial senescence and resulted in a significant reduction in aortic arch plaque area in HFD-fed ApoeKO mice. Consistently, specific deficiency or pharmacological inhibition of STING attenuated OSS-induced senescence and endothelial dysfunction. Pharmacological depletion of mtDNA ameliorated OSS-induced senescence and endothelial dysfunction. Taken together, our study linked hemodynamics and endothelial senescence, and revealed a novel mechanism by which OSS leads to endothelial dysfunction. Our study provided new insights into the development of therapeutic strategies for endothelial senescence and atherosclerosis.

The effect of underwater massage during hot water immersion on acute cardiovascular and mood responses

PurposeThere is emerging evidence that demonstrates the health benefits of hot water immersion including improvements to cardiovascular health and reductions in stress and anxiety. Many commercially available hot tubs offer underwater massage systems which purport to enhance many benefits of hot water immersion, however, these claims have yet to be studied. MethodsTwenty participants (4 females) completed three, 30-min sessions of hot-water immersion (beginning at 39 °C) in a crossover randomized design: with air massage (Air Jet), water massage (Hydro Jet) or no massage (Control). Cardiovascular responses comprising; heart rate, blood pressure and superficial femoral artery blood flow and shear rate were measured. State trait anxiety, basic affect, and salivary cortisol were recorded before and after each trial. Data were analysed using a mixed effects model. ResultsPost immersion, heart rate increased (Δ31bpm, P < 0.001, d = 1.38), mean arterial blood pressure decreased (Δ16 mmHg, P < 0.001, d = −0.66), with no difference between conditions. Blood flow and mean shear rate increased following immersion (P < 0.001, Δ362 ml/min, d = 1.20 and Δ108 s−1, d = 1.00), but these increases were blunted in the Air Jet condition (P < 0.001,Δ171 ml/min, d = 0.43 and Δ52 s−1, d = 0.52). Anxiety and salivary cortisol were reduced (P = 0.003, d = −0.20, P = 0.014, d = −0.11), but did not vary between conditions. Enjoyment did not vary between conditions. ConclusionThese data demonstrate positive acute responses to hot water immersion on markers of cardiovascular function, anxiety, and stress. There was no additional benefit of water-based massage, while air-based massage blunted some positive vascular responses due to lower heat conservation of the water.

Coronary hemodynamic simulation study.

In this paper, a two-way fluid-structure coupling model is developed to simulate and analyze the hemodynamic process based on dynamic coronary angiography, and examine the influence of different hemodynamic parameters on coronary arteries in typical coronary stenosis lesions. Using the measured FFR pressure data of a patient, the pressure-time function curve is fitted to ensure the accuracy of the boundary conditions. The average error of the simulation pressure results compared to the test data is 6.74%. In addition, the results related to blood flow, pressure contour and wall shear stress contour in a typical cardiac cycle are obtained by simulation analysis. These results are found to be in good agreement with the laws of the real cardiac cycle, which verifies the rationality of the simulation. In conclusion, based on the modeling and hemodynamic simulation analysis process of dynamic coronary angiography, this paper proposes a method to assist the analysis and evaluation of coronary hemodynamic and functional parameters, which has certain practical significance.

Epigenetic changes in shear-stressed endothelial cells.

Epigenetic changes, particularly histone compaction modifications, have emerged as critical regulators in the epigenetic pathway driving endothelial cell phenotype under constant exposure to laminar forces induced by blood flow. However, the underlying epigenetic mechanisms governing endothelial cell behavior in this context remain poorly understood. To address this knowledge gap, we conducted in vitro experiments using human umbilical vein endothelial cells subjected to various tensional forces simulating pathophysiological blood flow shear stress conditions, ranging from normotensive to hypertensive forces. Our study uncovers a noteworthy observation wherein endothelial cells exposed to high shear stress demonstrate a decrease in the epigenetic marks H3K4ac and H3K27ac, accompanied by significant alterations in the levels of HDAC (histone deacetylase) proteins. Moreover, we demonstrate a negative regulatory effect of increased shear stress on HOXA13 gene expression and a concomitant increase in the expression of the long noncoding RNA, HOTTIP, suggesting a direct association with the suppression of HOXA13. Collectively, these findings represent the first evidence of the role of histone-related epigenetic modifications in modulating chromatin compaction during mechanosignaling of endothelial cells in response to elevated shear stress forces. Additionally, our results highlight the importance of understanding the physiological role of HOXA13 in vascular biology and hypertensive patients, emphasizing the potential for developing small molecules to modulate its activity. These findings warrant further preclinical investigations and open new avenues for therapeutic interventions targeting epigenetic mechanisms in hypertensive conditions.

Predicting the Pharmacological Targets of Astragalus membranaceus against Hypertensive Nephropathy

Objective: Hypertension is one of the main causes of chronic kidney disease. Astragalus membranaceus (AM), an important traditional Chinese medicine for treating hypertensive nephropathy, has a complex composition that makes it challenging to explore its mechanism of action and limits its clinical application. This study aims to investigate the underlying mechanism of AM in treating hypertensive nephropathy. Methods: We retrieved all the compound data of AM from the Traditional Chinese Medicine Systems Pharmacology database and screened out the active compounds and their target proteins. Then, a network of candidate compounds and target compounds of AM was constructed using Cytoscape software. Furthermore, hypertensive nephropathy-related genes from the DisGeNET and GeneCards databases were intersected with AM target proteins and hypertensive nephropathy-related genes to determine the potential targets of AM in treating hypertensive nephropathy. Finally, after performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, we conducted molecular docking to verify the interaction between the main active ingredients of AM and the core targets. Results: A total of 87 effective components of AM were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. According to the network of active compounds and their target proteins, 18 of the 20 effective compounds in AM could act on 210 proteins. Taking the intersection of 274 hypertensive nephropathy-related genes and AM target proteins, 49 potential targets of AM in treating hypertensive nephropathy were identified. Using the median degree value, we determined 25 core targets of AM in treating hypertensive nephropathy. GO enrichment analysis showed that the biological processes of AM on hypertensive nephropathy mainly focused on the inflammatory response, hypoxia response, angiogenesis, cell proliferation, and cell migration. KEGG pathway enrichment analysis mainly involved cancer pathways, the AGE-RAGE signaling pathway in diabetic complications, blood flow shear stress, and atherosclerosis. Molecular docking results showed that quercetin, kaempferol, and 7-O-methylisomucronulatol had strong binding activity with several target proteins and may exert protective effects by stabilizing the interaction between molecules through the intermolecular forces of hydrogen bonds. Conclusion: This study reveals the targets of AM in treating hypertensive nephropathy using network pharmacology and molecular docking, providing new clues for developing novel drugs for hypertensive nephropathy and basic research development.

Self-Reinforced PTLG Copolymer with Shish Kebab Structures and a Bionic Surface as Bioimplant Materials for Tissue Engineering Applications.

Green and biodegradable materials with great mechanical properties and biocompatibility will offer new opportunities for next-generation high-performance biological materials. Herein, the novel oriented shish kebab crystals of a novel poly(trimethylene carbonate-lactide-glycolide) (PTLG) vascular stent are first reported to be successfully fabricated through a feasible solid-state drawing process to simultaneously enhance the mechanical performance and biocompatibility. The crystal structure of this self-reinforced vascular stent was transformed from spherulites to a shish kebab crystal, which indicates the mechanical interlocking effect and prevents the lamellae from slipping with a significant improvement of mechanical strength to 333 MPa. Meanwhile, it is different from typical biomedical polymers with smooth surface structures, and the as-obtained PTLG vascular stent exhibits a bionic surface morphology with a parallel micro groove and ridge structure. These ridges and grooves were attributed to the reorganization of cytoskeleton fiber bundles following the direction of blood flow shear stress. The structure and parameters of these morphologies were highly similar to the inner surface of blood vessels of the human, which facilitates cell adhesion growth to improve its proliferation, differentiation, and activity on the surface of PTLG.

Intravital two-photon microscopy of the native mouse thymus.

The thymus, a key organ in the adaptive immune system, is sensitive to a variety of insults including cytotoxic preconditioning, which leads to atrophy, compression of the blood vascular system, and alterations in hemodynamics. Although the thymus has innate regenerative capabilities, the production of T cells relies on the trafficking of lymphoid progenitors from the bone marrow through the altered thymic blood vascular system. Our understanding of thymic blood vascular hemodynamics is limited due to technical challenges associated with accessing the native thymus in live mice. To overcome this challenge, we developed an intravital two-photon imaging method to visualize the native thymus in vivo and investigated functional changes to the vascular system following sublethal irradiation. We quantified blood flow velocity and shear rate in cortical blood vessels and identified a subtle but significant increase in vessel leakage and diameter ~24 hrs post-sublethal irradiation. Ex vivo whole organ imaging of optically cleared thymus lobes confirmed a disruption of the thymus vascular structure, resulting in an increase in blood vessel diameter and vessel area, and concurrent thymic atrophy. This novel two-photon intravital imaging method enables a new paradigm for directly investigating the thymic microenvironment in vivo.

Monitoring of Hemostatic Status and Treatment Response in Von Willebrand Disease Using a Microchip Flow Chamber Assay

Introduction: Von Willebrand factor (VWF) carries out its hemostatic roles by interacting with both platelets and FVIII, and its appropriate active multimeric conformation is strongly affected by blood flow shear. Our aim was to assess the hemostatic status and the response to treatments in VW disease (VWD) using a flow chamber-based thrombus formation analysis system (T-TAS ®, Zacros) that allows evaluation of both primary and secondary hemostasis in a scenario closer to in vivo conditions. Methods: Blood samples from patients diagnosed with VWD type 1 (n=3), type 2A (n=2), type 2B (n=1) and type 3 (n=8, two of them with inhibitory antibodies) were collected before and after in vivo administration of the prescribed treatment with desmopressin (DDAVP ®) or plasma-derived (pd)VWF/FVIII concentrates (Table-1). The effect of ex vivo administration of 100, 150 and 300 IU/dL of pdVWF/FVIII (Fanhdi ®, Grifols) was also evaluated. Thrombus formation analysis was performed according to the manufacturer's standardized protocols: Blood samples collected in tubes containing BAPA (inhibitor of FXa and thrombin) were loaded onto type-1 collagen-coated platelet (PL)-chips to assess platelet-dependent thrombus formation. Citrated blood samples were recalcified in presence of CTI and loaded onto collagen/tissue factor-coated atherome (AR)-chips to assess thrombus formation mediated by both platelet and coagulation activation. An optimized flow rate is automatically adjusted for each chip. Area under the flow-pressure curve (AUC) values were collected. Platelet count, fibrinogen levels, prothrombin time (PT), activated partial thromboplastin time (aPTT), aPTT ratio, antigenic VWF (VWF:Ag), VWF activity by recombinant GPIb binding assay (VWF:GPIbR) and FVIII activity (FVIII:C) were also analyzed. Results: All VWD patient samples tested, including type-1 patients, showed a deficient platelet function (PL_AUC) and a reduced coagulation-dependent thrombus formation (AR_AUC) prior to treatment (Table-1). VWD type-1 responded to desmopressin with a high increase of FVIII activity (FVIII:C &amp;gt;200%) and normalization of VWF activity (VWF:GPIbR &amp;gt;100%) and AUC levels. Treatment with pdVWF/FVIII increased VWF:GPIbR levels (&amp;gt;60% after treatment) in all cases, but failed to completely normalize the AUC values, which showed a slight increase (Table-1). A high correlation (r&amp;gt;0.8) was observed between plasma levels of VWF or FVIII activity and AUC values obtained both before and after treatment Ex vivo administration of pdVWF/FVIIIa resulted in a concentration-dependent increased of PL_ and AR_AUC, with values within the normal range at the highest dose tested (300 IU/dL, equivalent to 150 IU/kg) in all cases, except for VWD type-3 samples with inhibitor and for VWD type-2B sample with PL-chip assay (Figure-1). Conclusions: Monitoring treatment efficacy in VWD using a flow chamber-based thrombus formation analysis system could be a reliable and standardized method to assess the response to desmopressin and pdVWF/FVIII concentrates in patients with VWD, allowing an optimal personalized therapeutic dosing and a better prevention of bleeding episodes in these patients. Funding: Grifols; Zacros; Fundación Rodríguez Pascual; Instituto de Salud Carlos III (ISCIII) (PI22/01461), co-funded by the European Union.

Enhanced external counterpulsation treatment regulates blood flow and wall shear stress metrics in femoral artery: An in vivo study in healthy subjects

As a non-invasive assisted circulation therapy, enhanced external counterpulsation (EECP) has demonstrated potential in treatment of lower-extremity arterial disease (LEAD). However, the underlying hemodynamic mechanism remains unclear. This study aimed to conduct the first prospective investigation of the EECP-induced responses of blood flow behavior and wall shear stress (WSS) metrics in the femoral artery. Twelve healthy male volunteers were enrolled. A Doppler ultrasound-basedapproach was introduced for the in vivo determination of blood flow in the common femoral artery (CFA) and superficial femoral artery (SFA) during EECP intervention, with incremental treatment pressures ranging from 10 to 40 kPa. Three-dimensional subject-specific numerical models were developed in 6 subjects to quantitatively assess variations in WSS-derived hemodynamic metrics in the femoral bifurcation. A mesh-independence analysis was performed. Our results indicated that, compared to the pre-EECP condition, both the antegrade and retrograde blood flow volumes in the CFA and SFA were significantly augmented during EECP intervention, while the heart rate remained constant. The time average shear stress (TAWSS) over the entire femoral bifurcation increased by 32.41%, 121.30%, 178.24%, and 214.81% during EECP with treatment pressures of 10 kPa, 20 kPa, 30 kPa, and 40 kPa, respectively. The mean relative resident time (RRT) decreased by 24.53%, 61.01%, 69.81%, and 77.99%, respectively. The percentage of area with low TAWSS in the femoral artery dropped to nearly zero during EECP with a treatment pressure greater than or equal to 30 kPa. We suggest that EECP is an effective and non-invasive approach for regulating blood flow and WSS in lower extremity arteries.

Modulation of red blood cell nitric oxide synthase phosphorylation in the quiescent and exercising human forearm.

In vitro investigations demonstrate that human erythrocytes synthesize nitric oxide via a functional isoform of endothelial nitric oxide synthase (NOS) (RBC-NOS). We tested the hypothesis that phosphorylation of RBC-NOS at serine residue 1177 (RBC-NOS1177) would be amplified in blood draining-active skeletal muscle. Furthermore, given hypoxemia modulates local blood flow and thus shear stress, and nitric oxide availability, we performed duplicate experiments under normoxia and hypoxia. Nine healthy volunteers performed rhythmic handgrip exercise at 60% of individualized maximal workload for 3.5 min while breathing room air (normoxia) and after being titrated to an arterial oxygen saturation ≈80% (hypoxemia). We measured brachial artery blood flow by high-resolution duplex ultrasound, while continuously monitoring vascular conductance and mean arterial pressure using finger photoplethysmography. Blood was sampled during the final 30 s of each stage from an indwelling cannula. Blood viscosity was measured to facilitate calculation of accurate shear stresses. Erythrocytes were assessed for levels of phosphorylated RBC-NOS1177 and cellular deformability from blood collected at rest and during exercise. Forearm exercise increased blood flow, vascular conductance, and vascular shear stress, which coincided with a 2.7 ± 0.6-fold increase in RBC-NOS1177 phosphorylation (P < 0.0001) and increased cellular deformability (P < 0.0001) under normoxia. When compared with normoxia, hypoxemia elevated vascular conductance and shear stress (P < 0.05) at rest, while cellular deformability (P < 0.01) and RBC-NOS1177 phosphorylation (P < 0.01) increased. Hypoxemic exercise elicited further increases in vascular conductance, shear stress, and cell deformability (P < 0.0001), although a subject-specific response in RBC-NOS1177 phosphorylation was observed. Our data yield novel insights into the manner that hemodynamic force and oxygen tension modulate RBC-NOS in vivo.

Image-based flow simulation of platelet aggregates under different shear rates.

Hemodynamics is crucial for the activation and aggregation of platelets in response to flow-induced shear. In this paper, a novel image-based computational model simulating blood flow through and around platelet aggregates is presented. The microstructure of aggregates was captured by two different modalities of microscopy images of in vitro whole blood perfusion experiments in microfluidic chambers coated with collagen. One set of images captured the geometry of the aggregate outline, while the other employed platelet labelling to infer the internal density. The platelet aggregates were modelled as a porous medium, the permeability of which was calculated with the Kozeny-Carman equation. The computational model was subsequently applied to study hemodynamics inside and around the platelet aggregates. The blood flow velocity, shear stress and kinetic force exerted on the aggregates were investigated and compared under 800 s-1, 1600 s-1 and 4000 s-1 wall shear rates. The advection-diffusion balance of agonist transport inside the platelet aggregates was also evaluated by local Péclet number. The findings show that the transport of agonists is not only affected by the shear rate but also significantly influenced by the microstructure of the aggregates. Moreover, large kinetic forces were found at the transition zone from shell to core of the aggregates, which could contribute to identifying the boundary between the shell and the core. The shear rate and the rate of elongation flow were investigated as well. The results imply that the emerging shapes of aggregates are highly correlated to the shear rate and the rate of elongation. The framework provides a way to incorporate the internal microstructure of the aggregates into the computational model and yields a better understanding of the hemodynamics and physiology of platelet aggregates, hence laying the foundation for predicting aggregation and deformation under different flow conditions.

Wall shear rate and energy loss coefficient measures using conventional Doppler ultrasound do not predict carotid plaque progression.

Background: The rate of carotid plaque progression is believed to be related to blood flow hemodynamics and shear stress. Our objective was to determine if wall shear rate (WSR) and the energy loss coefficient (ELC) measured by Doppler ultrasound could predict atherosclerotic carotid disease progression. Patients and methods: Patients at a large tertiary center with an initial ultrasound between 2016 and 2018 with a significant carotid plaque were included if they had at least one 6 months follow-up comparative study. Stenosis progression was assessed according to the NASCET (The North American Symptomatic Carotid Endarterectomy Trial) percentage criterion. Results: The average annual progression rate for the 74 plaques included was 5.7% NASCET per year. We identified 18 plaques with ≥10% NASCET progression and 56 plaques without significant progression <10% NASCET. Among the plaques with progression, only four plaques had progression greater than 20% NASCET. Median WSR was 6266 s-1 [5813-8974] in plaques with progression and 6564 s-1 [5285-8766] in stable plaques (p=0.643). Median ELC was 3.86 m2 [2.78-5.53] in plaque with progression and 4.32 m2 [3.42-6.81] in stable plaques (p=0.296). Conclusions: Although it is a widely accepted hypothesis that shear stress and hemodynamics of the carotid bifurcation contribute to plaque progression, we found that WSR and ELC estimated by Doppler ultrasound do not reliably predict atherosclerotic plaque progression in the carotid artery. Other ultrasound modalities, such as 3D imaging, may be used to assess the influence of plaque geometry and hemodynamics in plaque progression.