Mouse Carotid Artery Research Articles

Effects of Hydrogen Sulfide at Normal Body Temperature and in the Cold on Isolated Tail and Carotid Arteries from Rats and TRPA1 Knockout and Wild-Type Mice

Background: Hydrogen sulfide (H2S) is a gasotransmitter that modulates vascular tone, causing either vasodilation or vasoconstriction depending on the vascular bed, species, and experimental conditions. The cold-sensitive transient receptor potential ankyrin-1 (TRPA1) channel mediates H2S-induced effects; however, its contribution to the vasomotor responses of different arteries at different temperatures has remained unclear. Here, we aimed to fill this gap by comparing the effects of sodium sulfide (Na2S), which is a fast-releasing H2S donor, on the isolated carotid and tail skin arteries of rats and mice at cold and normal body temperature with wire myography. Under the same circumstances, we also aimed to compare the effects of the canonical endothelium-dependent and -independent vasodilators, acetylcholine and sodium nitroprusside, respectively. Methods: We isolated the carotid and tail arteries from 32 adult Wistar rats and 64 TRPA1 knockout and wild-type mice, and then we studied their vasomotor responses to increasing doses (10−6–10−3 M) of Na2S as well as to acetylcholine and sodium nitroprusside (10−5 M for both) at 37 °C and in cold (17 or 20 °C). Results: In rat vessels, Na2S caused constriction of the carotids and relaxation of the tail arteries, which were not influenced by temperature. In mouse carotids, Na2S caused vasorelaxation, which was more pronounced in the cold at a lower dose (10−4 M). At a higher dose (10−3 M), the dilation was markedly attenuated in the absence of the TRPA1 channel. In the mouse tail arteries, Na2S caused vasorelaxation at 37 °C and vasocontraction in the cold. The genetic blockade of TRPA1 channels did not influence the vasomotor responses of the mouse tail arteries. Sodium nitroprusside-induced vasorelaxation was not influenced by any of the investigated factors, while acetylcholine-induced dilation decreased in the cold in all vessel types. Conclusions: Our results reveal the function of TRPA1 in the H2S-induced dilation of carotid arteries in mice. We also highlight interspecies differences in the vasomotor responses between rats and mice, as well as the importance of the effect of temperature on vascular responses. The implementation of the identified variables in future research can advance our understanding of cardiovascular physiology, especially in conditions with hypothermia (either accidental or therapeutic).

Xiyangshen Sanqi Danshen granules attenuated D-gal-induced C57BL/6J mouse aging through the AMPK/SIRT1 signaling pathway

BackgroundAging is a pressing global concern and is frequently accompanied by the emergence of many chronic diseases. Xiyangshen Sanqi Danshen granules (XSD) have antioxidant, anti-inflammatory and anti-fatigue functions, but the mechanism of their anti-aging effects is not clear. MethodsThis study elucidated the anti-aging mechanism and potentially active ingredients of XSD by performing transcriptomic analysis and network pharmacological analysis in a D-galactose (D-gal)-induced C57BL/6J mouse aging model. ResultsXSD improved learning and memory abilities while enhanced motor function in D-gal-induced aging mice, as shown by Morris water maze, passive avoidance test, and rotating rod test results. Additionally, XSD significantly increased the vascular pulse wave velocity (PWV), β-stiffness index and pressure strain elastic coefficient (EP), decreased carotid distensibility (CD) and decreased the expression levels of P53 and 8-OHdG in the common carotid arteries of D-gal mice. Transcriptome sequencing analysis identified that the AMPK/SIRT1 signaling pathway is the potential mechanism by which XSD attenuates aging. XSD also increased the protein levels of Ki67, AMPK, SIRT1 and the nuclear translocation of Nrf2 while decreased the protein levels of P21, P53, IL-18, 8-OHdG, nitrotyrosine, and COX-2 and the nuclear translocation of NF-κB p65 in the brains of D-gal-induced mice. The administration of the AMPK inhibitor and SIRT1 inhibitor hindered the anti-aging effect of XSD, as indicated by an elevation of 8-OHdG, COX-2, and nuclear translocation of NF-κB p65 ; and a decrease of Ki67 and the nuclear translocation of Nrf2. Network pharmacological analysis revealed that the potential active ingredients of XSD were quercetin, kaempferol, tanshinone IIA, isorhamnetin, ginsenoside F2, and cryptotanshinone. ConclusionCollectively, XSD mitigated D-gal-induced aging in C57BL/6J mice through enhancing the AMPK/SIRT1 signaling pathway. This research provides potential drugs for anti-aging and also promotes the usage of the anti-aging effect of XSD.

Shear-Sensing by C-Reactive Protein: Linking Aortic Stenosis and Inflammation.

CRP (C-reactive protein) is a prototypical acute phase reactant. Upon dissociation of the pentameric isoform (pCRP [pentameric CRP]) into its monomeric subunits (mCRP [monomeric CRP]), it exhibits prothrombotic and proinflammatory activity. Pathophysiological shear rates as observed in aortic valve stenosis (AS) can influence protein conformation and function as observed with vWF (von Willebrand factor). Given the proinflammatory function of dissociated CRP and the important role of inflammation in the pathogenesis of AS, we investigated whether shear stress can modify CRP conformation and induce inflammatory effects relevant to AS. To determine the effects of pathological shear rates on the function of human CRP, pCRP was subjected to pathophysiologically relevant shear rates and analyzed using biophysical and biochemical methods. To investigate the effect of shear on CRP conformation in vivo, we used a mouse model of arterial stenosis. Levels of mCRP and pCRP were measured in patients with severe AS pre- and post-transcatheter aortic valve implantation, and the presence of CRP was investigated on excised valves from patients undergoing aortic valve replacement surgery for severe AS. Microfluidic models of AS were then used to recapitulate the shear rates of patients with AS and to investigate this shear-dependent dissociation of pCRP and its inflammatory function. Exposed to high shear rates, pCRP dissociates into its proinflammatory monomers (mCRP) and aggregates into large particles. Our in vitro findings were further confirmed in a mouse carotid artery stenosis model, where the administration of human pCRP led to the deposition of mCRP poststenosis. Patients undergoing transcatheter aortic valve implantation demonstrated significantly higher mCRP bound to circulating microvesicles pre-transcatheter aortic valve implantation compared with post-transcatheter aortic valve implantation. Excised human stenotic aortic valves display mCRP deposition. pCRP dissociated in a microfluidic model of AS and induces endothelial cell activation as measured by increased ICAM-1 (intercellular adhesion molecule 1) and P-selectin expression. mCRP also induces platelet activation and TGF-β (transforming growth factor beta) expression on platelets. We identify a novel mechanism of shear-induced pCRP dissociation, which results in the activation of cells central to the development of AS. This novel mechanosensing mechanism of pCRP dissociation to mCRP is likely also relevant to other pathologies involving increased shear rates, such as in atherosclerotic and injured arteries.

KIF11 promotes vascular smooth muscle cell proliferation by regulating cell cycle progression and accelerates neointimal formation after arterial injury in mice.

Background and aims: One of the primary causes of lumen narrowing is vascular injury induced during medical procedures. Vascular injury disrupts the integrity of the endothelium, triggering platelet deposition, leukocyte recruitment, and the release of inflammatory factors. This, in turn, induces the proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation. However, the molecular mechanism underlying VSMC proliferation following injury remains unknown. KIF11 is critical in regulating the cell cycle by forming bipolar spindles during mitotic metaphase. This process may contribute to VSMCs proliferation and neointima formation following vascular injury. Yet, the function of KIF11 in VSMCs has not been elucidated. This study aims to investigate the role and mechanisms of KIF11 in regulating VSMCs cycle progression and proliferation. Methods: After conducting biological analysis of the transcriptome sequencing data from the mouse carotid artery injury model and the cell transcriptome data of PDGF-BB-induced VSMCs, we identified a potential target gene, KIF11, which may play a crucial role in vascular injury. Then we established a vascular injury model to investigate how changes in KIF11 expression and activity influence in vivo VSMCs proliferation and neointimal formation. In addition, we employed siRNA and specific inhibitors to suppress KIF11 expression and activity in VSMCs cultured in vitro to study the mechanisms underlying VSMCs cycle progression and proliferation. Results: The results of immunohistochemistry and immunofluorescence indicate a significant upregulation of KIF11 expression in the injured vascular. The intraperitoneal injection of the KIF11 specific inhibitor, K858, partially inhibits intimal hyperplasia in the vascular injury model. In vitro experiments further demonstrate that PDGF-BB upregulates KIF11 expression through the PI3K/AKT pathway, and enhances KIF11 activity. Inhibition of both KIF11 expression and activity partially reverses the pro-cycle progression and pro-proliferation effects of PDGF-BB on VSMCs. Additionally, KIF11 overexpression partially counteracts the proliferation arrest and cell cycle arrest induced by inhibiting the PI3K/AKT pathway in VSMCs. Conclusion: Our study highlights the crucial role of KIF11 in regulating the cycle progression and proliferation of VSMCs after vascular injury. A comprehensive understanding of these mechanisms could pave the way for potential therapeutic interventions in treating vascular stenosis.

The bottlenose dolphin (Tursiops truncatus): a novel model for studying healthy arterial aging.

Endothelial function declines with aging and independently predicts future cardiovascular disease (CVD) events. Diving also impairs endothelial function in humans. Yet, dolphins, being long-lived mammals adapted to diving, undergo repetitive cycles of tissue hypoxia-reoxygenation and disturbed shear stress without manifesting any apparent detrimental effects, as CVD is essentially nonexistent in these animals. Thus, dolphins may be a unique model of healthy arterial aging and may provide insights into strategies for clinical medicine. Emerging evidence shows that the circulating milieu (bioactive factors in the blood) is at least partially responsible for transducing reductions in age-related endothelial function. To assess whether dolphins have preserved endothelial function with aging because of a protected circulating milieu, we tested if the serum (pool of the circulating milieu) of bottlenose dolphins (Tursiops truncatus) induces the same arterial aging phenotype as the serum of age-equivalent humans. We incubated conduit arteries from young and old mice with dolphin and human serum and measured endothelial function ex vivo via endothelium-dependent dilation to acetylcholine. Although young arteries incubated with serum from midlife/older adult human serum had lower endothelial function, those incubated with dolphin serum consistently maintained high endothelial function regardless of the age of the donor. Thus, studying the arterial health of dolphins could lead to potential novel therapeutic strategies to improve age-related endothelial dysfunction in humans.NEW & NOTEWORTHY We demonstrate that, unlike serum of midlife/older adult humans, age-matched dolphin serum elicits higher endothelial function ex vivo in young mouse carotid arteries, suggesting that the circulating milieu of bottlenose dolphins may be geroprotective. We propose that dolphins are a novel model to investigate potential novel therapeutic strategies to mitigate age-related endothelial dysfunction in humans.

Intervention of Asprosin Attenuates Oxidative Stress and Neointima Formation in Vascular Injury.

Aims: Asprosin, a newly discovered hormone, is linked to insulin resistance. This study shows the roles of asprosin in vascular smooth muscle cell (VSMC) proliferation, migration, oxidative stress, and neointima formation of vascular injury. Methods: Mouse aortic VSMCs were cultured, and platelet-derived growth factor-BB (PDGF-BB) was used to induce oxidative stress, proliferation, and migration in VSMCs. Vascular injury was induced by repeatedly moving a guidewire in the lumen of the carotid artery in mice. Results: Asprosin overexpression promoted VSMC oxidative stress, proliferation, and migration, which were attenuated by toll-like receptor 4 (TLR4) knockdown, antioxidant (N-Acetylcysteine, NAC), NADPH oxidase 1 (NOX1) inhibitor ML171, or NOX2 inhibitor GSK2795039. Asprosin overexpression increased NOX1/2 expressions, whereas asprosin knockdown increased heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1) expressions. Asprosin inhibited nuclear factor E2-related factor 2 (Nrf2) nuclear translocation. Nrf2 activator sulforaphane increased HO-1 and NQO-1 expressions and prevented asprosin-induced NOX1/2 upregulation, oxidative stress, proliferation, and migration. Exogenous asprosin protein had similar roles to asprosin overexpression. PDGF-BB increased asprosin expressions. PDGF-BB-induced oxidative stress, proliferation, and migration were enhanced by Nrf2 inhibitor ML385 but attenuated by asprosin knockdown. Vascular injury increased asprosin expression. Local asprosin knockdown in the injured carotid artery promoted HO-1 and NQO-1 expressions but attenuated the NOX1 and NOX2 upregulation, oxidative stress, neointima formation, and vascular remodeling in mice. Innovation and Conclusion: Asprosin promotes oxidative stress, proliferation, and migration of VSMCs via TLR4-Nrf2-mediated redox imbalance. Inhibition of asprosin expression attenuates VSMC proliferation and migration, oxidative stress, and neointima formation in the injured artery. Asprosin might be a promising therapeutic target for vascular injury. Antioxid. Redox Signal. 41, 488-504.

The bottlenose dolphin (Tursiops truncatus): A novel model for studying healthy vascular aging

Aging is the primary non-modifiable risk factor for the development of cardiovascular diseases (CVD). CVD are often preceded by arterial dysfunction, including endothelial dysfunction and aortic stiffening. Our preliminary results suggest that the circulating milieu is associated with differences in arterial function in young and mid-life/older (ML/O) adults. Moreover, incubation of mouse arteries with serum from young and ML/O adult humans can directly transfer arterial aging phenotypes for endothelial function and aortic stiffness. These results suggest that age-related changes in the circulating milieu are an underlying mechanism of age-related arterial dysfunction. Dolphins are mammals that have evolutionary adapted to diving, with constant cycles of tissue hypoxia/reoxygenation and disturbed shear stress -- exposures which cause arterial dysfunction in humans. Dolphins also have long lifespans and are relatively free of CVD. Taken together, dolphins may be a unique model of healthy vascular aging, with possible applications to clinical medicine. PURPOSE: To determine if the serum of bottlenose dolphins ( Tursiops truncatus) induces the same arterial aging phenotypes as the serum of age-equivalent humans. Methods: Common carotid arteries and aorta rings from young (Y; 5 mo) and old (O; 25 mo) wildtype female (F) and male (M) C57BL/6N mice were exposed ex vivo for 24 and 48h, respectively, to 5% sex-matched adult dolphin serum. Dolphin serum of Y (17 ± 4 years; n = 16: 5F / 11 M) and ML/O (29 ± 2 years; n = 4: 1F/3M) adults was obtained from the National Marine Mammal Tissue Bank. Endothelial function was assessed as carotid artery endothelium-dependent dilation (EDD) in response to increasing doses of acetylcholine. Aortic stiffness was measured as elastic modulus, a measure of intrinsic mechanical wall stiffness. Dolphin serum-mediated responses in mouse arteries were compared with that of adult human serum data (Y, 24 ± 1 years, n=10: 5F/5M; ML/O, 67 ± 3 years, n=10: 5F/5M). Differences were assessed via 2-way ANOVAs; within species post hoc analyses conducted between age groups. Results: Endothelial Function. Peak EDD in Y mouse carotid arteries was lower following exposure to serum from ML/O adult humans (ML/O, 78 ± 2 v. Y, 92 ± 2%; P < 0.001) and higher in O mouse carotid arteries after exposure to Y adult serum (92 ± 2 vs. 73 ± 5%, P = 0.001). In contrast, EDD was similarly high across all groups following dolphin serum exposure (³ 93% peak EDD), independent of the age of the serum donor or the mouse arteries (Y v. ML/O serum donor in Y arteries, P = 0.773; in O mouse arteries, P = 0.942). Aortic Stiffness. Stiffness in Y aortic rings was ~60% higher following exposure to ML/O adult human serum (P = 0.001 v. Y adult serum). In O mouse aortic rings, stiffness was 25% lower after exposure to Y adult serum (P = 0.044 v. ML/O adult serum). In contrast, stiffness was similar across all groups following dolphin serum exposure, independent of age of the serum donor or the arteries (Y v. ML/O serum donor in Y aortic rings, P = 0.974; in O aortic rings P = 0.157). CONCLUSIONS: The circulating milieu of bottlenose dolphins may be geroprotective, which could serve as a model to investigate mechanisms and potential therapies for preventing adverse vascular aging and promoting CV health and longevity in humans. FUNDING: R21 AG078408 & MSCA-IF-2019:892267. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 110: Pro-thrombotic BCR-ABL Tyrosine Kinase Inhibitors Ponatinib And Nilotinib, But Not The Novel Agent Asciminb, Impair Endothelial Cell Wound Healing

Introduction: Tyrosine kinase inhibitors (TKIs) targeting ABL kinase have transformed the treatment of BCR-ABL positive leukemias. First generation imatinib is well tolerated while the newer TKIs, ponatinib and nilotinib, are more effective for leukemia but both substantially increase risk of acute arterial thrombosis, thereby limiting their clinical benefit over imatinib. Asciminib is a new BCR-ABL inhibitor with an unknown safety profile. We previously showed that BCR-ABL inhibitors that increase thrombosis in humans also cause toxicity in endothelial cells (ECs). Hypothesis: We hypothesize that BCR-ABL TKIs that cause EC dysfunction in vitro delay healing at sites of vascular injury in vivo as a potential mechanism of increased risk of thrombosis. Methods: We used the in vitro scratch wound assay and western blot for endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein (HUV)ECs and a mouse carotid artery wire injury model to assess the impact of BCR-ABL TKIs on EC wound healing capacity in vitro and in vivo . Results: ECs were treated with the Cmax concentration in cancer patients, as were mice, as confirmed by serum LC-MS. In vitro , ponatinib and nilotinib significantly decreased HUVEC wound healing compared to both imatinib and asciminib (p<0.001), with no difference between imatinib and asciminib. Ponatinib significantly decreased peNOS compared to both imatinib and nilotinib (<0.05), while asciminib was not significantly different from any of the other TKIs. In vivo , nilotinib significantly decreased carotid EC wound healing compared to imatinib and asciminib (p< 0.05), while ponatinib was not significantly different from any other TKIs. Carotid thrombosis after wire injury was observed in 3/26 mice treated with ponatinib, 5/23 with nilotinib, 1/21 with asciminib, and 0/26 treated with imatinib, with clot-free survival studies ongoing. Conclusions: Ponatinib and nilotinib cause EC toxicity by distinct mechanisms in vitro , which has implications for mitigating their cardiovascular risk. Asciminib causes little vascular toxicity in vitro or in vivo by these assays. Carotid thrombosis developed in some TKI treated mice, suggesting this model can be used to study the link between impaired EC healing and thrombosis.

Numerical Analysis of Mice Carotid Arteries’ Response Emphasizing the Importance of Material Law Constants’ Validation

In this paper, a detailed validation of the passive material properties of mice carotid arteries and constants of the Fung and Holzapfel hyperelastic material laws is conducted by means of static nonlinear FEM analyses. The response of the carotid arteries in an inflation test is studied here for the following mouse models: wild-type, mdx, sgcd−/−, Eln+/+, Eln+/−, Fbln5+/+, and Fbln5−/−. All FEM computations are conducted on models that have been preliminarily checked for their reliability. The results of the calculations, namely, the relation between the internal pressure and the artery outer diameter, are verified against experimental responses and the applicability of the laws is assessed. New sets of Holzapfel constitutive relation constants are proposed for Eln+/+ and Fbln5−/− mice. Finally, the problem of carotid artery buckling is also discussed. The buckling pressures of the arteries are predicted using FEM models and nonlinear static analyses. These values are compared with the reference experimental results, which allow for further validation of the constitutive relations. The research emphasizes that computations and numerical methods enable an accurate description of bioengineering processes and behaviors but only if the models used are appropriately validated.

Tumor necrosis factor receptor-associated factor 5 protects against intimal hyperplasia by regulation of macrophage polarization via directly targeting PPARγ.

Intimal hyperplasia is a serious clinical problem associated with the failure of therapeutic methods in multiple atherosclerosis-related coronary heart diseases, which are initiated and aggravated by the polarization of infiltrating macrophages. The present study aimed to determine the effect and underlying mechanism by which tumor necrosis factor receptor-associated factor 5 (TRAF5) regulates macrophage polarization during intimal hyperplasia. TRAF5 expression was detected in mouse carotid arteries subjected to wire injury. Bone marrow-derived macrophages, mouse peritoneal macrophages and human myeloid leukemia mononuclear cells were also used to test the expression of TRAF5 in vitro. Bone marrow-derived macrophages upon to LPS or IL-4 stimulation were performed to examine the effect of TRAF5 on macrophage polarization. TRAF5-knockout mice were used to evaluate the effect of TRAF5 on intimal hyperplasia. TRAF5 expression gradually decreased during neointima formation in carotid arteries in a time-dependent manner. In addition, the results showed that TRAF5 expression was reduced in classically polarized macrophages (M1) subjected to LPS stimulation but was increased in alternatively polarized macrophages (M2) in response to IL-4 administration, and these changes were demonstrated in three different types of macrophages. An in vitro loss-of-function study with TRAF5 knockdown plasmids or TRAF5-knockout mice revealed high expression of markers associated with M1 macrophages and reduced expression of genes related to M2 macrophages. Subsequently, we incubated vascular smooth muscle cells with conditioned medium of polarized macrophages in which TRAF5 expression had been downregulated or ablated, which promoted the proliferation, migration and dedifferentiation of VSMCs. Mechanistically, TRAF5 knockdown inhibited the activation of anti-inflammatory M2 macrophages by directly inhibiting PPARγ expression. More importantly, TRAF5-deficient mice showed significantly aggressive intimal hyperplasia. Collectively, this evidence reveals an important role of TRAF5 in the development of intimal hyperplasia through the regulation of macrophage polarization, which provides a promising target for arterial restenosis-related disease management.

Abstract 4140: Enhanced microglial phagocytosis of metastatic brain tumor cells with Cd24a/Cd47 depletion

Abstract Despite the robust homeostasis of the brain, its high prevalence of tumor metastasis is intriguing and the underlying mechanism may contribute to the development of novel therapies. Microglia have emerged as promising therapeutic targets due to their role in brain parenchymal homeostasis. However, with evidence for both pro- and anti-tumorigenic functions, their role in brain metastasis (BrM) remains unclear. Here, we show that the microglial response to tumor cells within a single host is heterogeneous and can alter tumor fate. Single-cell RNA sequencing was performed on five surgically resected metastatic brain tumors, and the transcriptome of tumor-associated microglia was extracted by single-cell variational inference (scVI). Evidence for the coexistence of cytokine-secreting and angiogenesis-supporting subsets, as well as subsets associated with phagocytosis and antigen presentation, within each tumor sample was determined. To demonstrate the intra-tumoral functional heterogeneity of tumor-associated microglia, we established a metastatic brain tumor model that allows us to visualize the metastatic process and the dynamic microglial response in vivo by injecting mCherry-tagged allogeneic cancer cells into the internal carotid artery of CX3CR1-EGFP mice. Metastasis formation was suppressed in microglia-depleted mice, identifying a dominant microglial response that promotes tumor growth. However, continuous in vivo imaging of the BrM model revealed tumor-eradicating anti-tumor microglia coexisting with pro-tumor microglia in the same host. The transcriptome of anti-tumor microglia supported that they are enriched for phagocytosis, antigen processing and antigen presentation genes. The subset was identified in microglia extracted from human samples. We then examined the immune checkpoint of microglial phagocytosis in the tumor transcriptome and identified Cd24a and Cd47. Simultaneous deletion of both genes in cancer cells enhanced microglial phagocytosis, reduced tumor volume and improved survival, and rescue of both genes reversed these anti-tumor effects. The microglial dependency was addressed by microglial depletion experiments, and the anti-tumor effects were reproducible in nude mice lacking mature T cells. These results suggest that there are distinct immune cell subtypes that respond to lung tumor metastases in the brain and either promote tumor growth or inhibit metastasis through phagocytosis. Furthermore, anti-tumor activity in mouse models can be enhanced by genetic deletion of Cd24a and Cd47 in tumor cells, which regulate phagocytosis by microglia. Citation Format: Takahiro Tsuji, Haruka Hirose, Mariko Shindo, Rahadian Yudo Hartantyo, Yutaro Saito, Kazutaka Hosoya, Hiroshi Yoshida, Daisuke Kato, Akihiko Yoshizawa, Yoshiki Arakawa, Hiroaki Ozasa, Toyohiro Hirai, Hiroaki Wake. Enhanced microglial phagocytosis of metastatic brain tumor cells with Cd24a/Cd47 depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4140.

Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, andEDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 μM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 μM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (Mφ/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Blood shear stress during the cardiac cycle and endothelial cell orientation and polarity in the carotid artery of male and female mice.

Introduction: Blood flow produces fluid shear stress (SS), a frictional force parallel to the blood flow, on the endothelial cell (EC) layer of the lumen of the vessels. ECs themselves are sensitive to this frictional force in terms of directionality and intensity. The aim of this study was to determine the physiological shear stress value during the cardiac cycle and EC polarity and orientation from blood flow in healthy male and female mouse carotid artery. Methods: Experimentation is done on anesthetized male and female 8-week-old C5BL/6J mice. In vivo measurements of maximum blood velocity and vessel diameter in diastole and systole were performed on the right common carotid artery by Doppler ultrasound imaging. Blood viscosity (total and plasmatic) and hematocrit were determined on blood samples. For SS calculation, we developed a new method assuming heterogenous blood flow, i.e., a red cell central plug flow surrounded by a peripheral plasma sheath flow, and computing SS from vessel diameter and hemodynamical measurements (maximal blood velocity, hematocrit and plasmatic viscosity). Results: Results were compared with the classical method assuming a homogenous blood flow with constant apparent total blood viscosity. EC polarity and orientation were determined ex vivo on the carotid endothelium by confocal imaging after labeling of the EC nucleus and Golgi apparatus. Diastolic and systolic SS were 6 ± 2.5 Pa and 30 ± 6.5 Pa, respectively. Total blood and plasmatic viscosity was 4 ± 0.5 cP and 1.27 cP, respectively. ECs were polarized and significantly oriented against blood flow. No sex difference was identified.

Eriocalyxin B alleviated ischemic cerebral injury by limiting microglia-mediated excessive neuroinflammation in mice.

Excessive neuroinflammation mediated by microglia has a detrimental effect on the progression of ischemic stroke. Eriocalyxin B (EriB) was found with a neuroprotective effect in mice with Parkinson's disease via the suppression of microglial overactivation. This study aimed to investigate the roles of EriB in permanent middle cerebral artery occlusion (pMCAO) mice. The pMCAO was induced in the internal carotid artery of the mice by the intraluminal filament method, and EriB (10 mg/kg) was administered immediately after surgery by intraperitoneal injection. The behavior score, 2,3,5-triphenyltetrazole chloride staining, Nissl staining, TUNEL, immunohistochemistry, immunofluorescence, PCR, ELISA, and immunoblotting revealed that EriB administration reduced brain infarct and neuron death and ameliorated neuroinflammation and microglia overactivation in pMCAO mice, manifested by alterations of TUNEL-positive cell numbers, ionized calcium binding adaptor molecule 1 (Iba-1)-positive cell numbers, and expression of tumor necrosis factor-α, interleukin 6, IL-1β, inducible nitric oxide synthase, and arginase 1. In addition, EriB suppressed ischemia-induced activation of nuclear factor kappa B (NF-κB) signaling in the brain penumbra, suggesting the involvement of NF-κB in EriB function. In conclusion, EriB exerted anti-inflammatory effects in ischemia stroke by regulating the NF-κB signaling pathway, and this may provide insights into the neuroprotective effect of EriB in the treatment of ischemic stroke.

Visualization of Pulse-Wave Velocity on Arterial Wall of Mice Through High-Frequency Ultrafast Doppler Imaging.

Arterial pulse-wave velocity (PWV) is widely used in clinical applications to assess cardiovascular diseases. Ultrasound methods have been proposed for estimating regional PWV in human arteries. Furthermore, high-frequency ultrasound (HFUS) has been applied to perform preclinical small-animal PWV measurements; however, electrocardiogram (ECG)-gated retrospective imaging is required to achieve high-frame-rate imaging, which might be affected by arrhythmia-related problems. In this article, HFUS PWV mapping based on 40-MHz ultrafast HFUS imaging is proposed to visualize PWV on mouse carotid artery to measure arterial stiffness without ECG gating. In contrast to most other studies that used cross-correlation methods to detect arterial motion, ultrafast Doppler imaging was applied in this study to measure arterial wall velocity for PWV estimations. The performance of the proposed HFUS PWV mapping method was verified using a polyvinyl alcohol (PVA) phantom with various freeze-thaw cycles. Small-animal studies were then performed in wild-type (WT) mice and in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (for 16 and 24 weeks). The Young's modulus of the PVA phantom measured through HFUS PWV mapping was 15.3 ± 0.81, 20.8 ± 0.32, and 32.2 ± 1.11 kPa for three, four, and five freeze-thaw cycles, respectively, and the corresponding measurement biases (relative to theoretical values) were 1.59%, 6.41%, and 5.73%, respectively. In the mouse study, the average PWVs were 2.0 ± 0.26, 3.3 ± 0.45, and 4.1 ± 0.22 m/s for 16-week WT, 16-week ApoE KO, and 24-week ApoE KO mice, respectively. The PWVs of ApoE KO mice increased during the high-fat diet feeding period. HFUS PWV mapping was used to visualize the regional stiffness of mouse artery, and a histology confirmed that the plaque formation in the bifurcation region increased the regional PWV. All the results indicate that the proposed HFUS PWV mapping method is a convenient tool for investigating arterial properties in preclinical small-animal studies.

Severe arterial injury heals with a complex clonal structure involving a large fraction of surviving smooth muscle cells

Background and aimsSmooth muscle cell (SMC) lineage cells in atherosclerosis and flow cessation-induced neointima are oligoclonal, being recruited from a tiny fraction of medial SMCs that modulate and proliferate. The present study aimed to investigate the clonal structure of SMC lineage cells healing more severe arterial injury. MethodsArterial injury (wire, stretch, and partial ligation) was inflicted on the right carotid artery in mice with homozygous, SMC-restricted, stochastically recombining reporter transgenes that produced mosaic expression of 10 distinguishable fluorescent phenotypes for clonal tracking. Healed arteries and contra-lateral controls were analyzed after 3 weeks. Additional analysis of cell death and proliferation after injury was performed in wildtype mice. ResultsThe total number of SMC lineage cells in healed arteries was comparable to normal arteries but comprised significantly fewer fluorescent phenotypes. The population had a complex, intermixed, clonal structure. By statistical analysis of expected versus observed fractions of fluorescent phenotypes and visual inspection of coherent groups of same-colored cells, we concluded that >98% of SMC lineage cells in healed arteries belonged to a detectable clone, indicating that nearly all surviving SMCs after severe injury at some point undergo proliferation. This was consistent with serial observations in the first week after injury, which showed severe loss of medial cells followed by widespread proliferation. ConclusionsAfter severe arterial injury, many surviving SMCs proliferate to repair the media and form a neointima. This indicates that the fraction of medial SMCs that are mobilized to repair arteries increases with the level of injury.

Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles.

Introduction: Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. Methods: RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation. Results: Our results showed that treating RBCs at 6% hematocrit with 10µM yoda1 for 30min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. Conclusion: This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated in vivo under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.

SMYD2 regulates vascular smooth muscle cell phenotypic switching and intimal hyperplasia via interaction with myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using an SMC-specific SMYD2 knockout mouse model, we found that SMYD2 ablation in VSMCs exacerbated neointima formation after vascular injury in vivo. Conversely, SMYD2 overexpression inhibited VSMC proliferation and migration in vitro and attenuated arterial narrowing in injured vessels in mice. SMYD2 downregulation promoted VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulated VSMC contractile gene expression and suppressed VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacted with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism.

Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery.

Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7-14 days for the histological characterization of the metastases' pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan-cytokeratin and tomato lectin) features, which account for metastases' establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies.