Smooth Muscle Layer Research Articles

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm

BackgroundThe fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood.MethodsIn this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRβ, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib.ResultsSingle-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation.ConclusionOur findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Understanding Samaana Vaata in relation to the physiology of digestion

Ayurveda is a way of life governed by the principle that whatever there is in the universe is there in the body also. The concept of Tridosha is one such theory which decides the well-being and the diseased condition of a human being. That is, when in equilibrium leads to health and when vitiated, leads to disease. Vaata being one of the Tridosha leads the other two Dosha, Mala, Dhatus as the wind carries the clouds in the sky. There are five types of Vaata, i.e., Prana, Udana, Samana, Vyana and Apana. Samaana, is situated near Jatharagni and moves all over the Koshta. The function of Samaana Vaata can be corelated with functions such as Grahana, Pachana, Vivechana, Munchana. The myenteric plexus, or plexus of Auerbach, is located between the longitudinal and circular smooth muscle layers of the muscularis in gastrointestinal tract. The motor neurons of these plexus mostly control GI tract motility (movement), particularly the frequency and strength of contraction of the muscularis. This can be co-related to Karma of Samana Vata like Grahana, Munchana. The submucosal plexus, also known as Meissner’s plexus, is a network of autonomic motor nerve fibres located in the submucosa of the gastrointestinal tract. The motor neurons of the submucosal plexus supply the secretory cells of the mucosal epithelium, controlling the secretions and absorption of the organs of the gastrointestinal tract. This can be co-related to Karma of Samana Vaata like Pachana, Vivechana. Hence this article is aimed at further exploration of these anatomical and physiological aspects.

Protective effect of equol intake on bladder dysfunction in a rat model of bladder outlet obstruction.

This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB). Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde). Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline. Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO.

Knockdown of the SDHB Subunit Induces Mitochondrial Remodeling in Human Airway Smooth Muscle Cells

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that serves a dual role in mitochondrial respiration: catalyzing the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and transferring electrons from succinate to ubiquinone as complex II in the electron transport chain (ETC). SDH is composed of four subunits (SDHA-SDHD) and the loss of SDHB subunit, specifically, has been shown to increase oxidative stress. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone. In human airway smooth muscle (hASM) cells with siRNA-mediated knockdown of SDHB, the maximum velocity of the SDH reaction (SDHmax) is decreased. Moreover, SDHB-knockdown leads to an increase in mitochondrial volume density, indicating a role of SDH in mitochondrial remodeling. However, the impact of SDHB-knockdown on mitochondrial dynamics has not been explored. Therefore, we hypothesize that SDHB-knockdown promotes mitochondrial fragmentation as part of mitochondrial remodeling in hASM cells. To investigate this hypothesis, bronchiolar tissue samples were collected in female and male patients undergoing lung surgery with no current history of smoking or respiratory diseases, the smooth muscle layer was dissected and hASM cells were dissociated. The targeted knockdown of SDHB was achieved by siRNA-mediated knockdown. Mitochondria in hASM cells were labeled with MitoTracker Green (200 nM) and imaged in 3D with a Z optical slice of 0.5 μm using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. Optical slices were deconvolved to enhance contrast and the MitoTracker labeling was thresholded to create binary images, which were then reconstructed in 3D. From these images, the mitochondrial complexity index (MCI) was calculated to quantify the extent of fragmentation. To identify the signaling pathway involved in mitochondrial fragmentation, the expression and phosphorylation of dynamin-related protein 1 (DRP1) was quantified by Western blot. Mitochondrial fragmentation is associated with increased phosphorylation of DRP1 at Ser616 (pDRP1s616). Furthermore, mitophagy was assessed by determining the mitochondrial expression of PINK1 and the phosphorylation of Parkin at Ser65 (pParkinS65). Mitochondrial biogenesis was also evaluated through the expression of PGC1α, NRF1/2 and TFAM. Our results show that SDHB-knockdown increased mitochondrial fragmentation (reduced MCI) in hASM cells, and increased pDRP1s616 phosphorylation and its translocation to mitochondria. SDHB-knockdown also increased mitochondrial expression of PINK1 and pParkins65 phosphorylation indicating activation of mitophagy. We also confirmed that SDHB-knockdown increased expression of PGC1α, NRF1/2 and TFAM consistent with mitochondrial biogenesis. These findings unveil the mechanisms underlying SDHB-knockdown-mediated mitochondrial remodeling. Supported by NIH grant R01-HL157984 (GCS). 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.

Improved Preservation of Mouse Intestinal Tissue Using a Formalin/Acetic Acid Fixative and Quantitative Histological Analysis Using QuPath.

The architecture and morphology of the intestinal tissue from mice or other small animals are difficult to preserve for histological and molecular analysis due to the fragile nature of this tissue. The intestinal mucosa consists of villi and crypts lined with epithelial cells. In between the epithelial folds extends the lamina propria, a loose connective tissue that contains blood and lymph vessels, fibroblasts, and immune cells. Underneath the mucosa are two layers of contractile smooth muscle and nerves. The tissue experiencessignificant changes during fixation, which can impair the reliability of histologic analysis. Poor-quality histologic sections are not suitable for quantitative image-based tissue analysis. This article offers a new fixative composed of neutral buffered formalin (NBF) and acetic acid, called FA. This fixative significantly improved the histology of mouse intestinal tissue compared to traditional NBF and enabled precise, reproducible histologic molecular analyses using QuPath software. Algorithmic training of QuPath allows for automated segmentation of intestinal compartments, which can be further interrogated for cellular composition and disease-related changes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Improved preservation of mouse intestinal tissue using a formalin/acetic acid fixative Support Protocol: Quantitative tissue analysis using QuPath.

Piezo1-dependent inhibition of background potassium current in the rat bladder smooth muscle cells

The smooth muscle layer of the urinal bladder (detrusor), along with the urothelium, has autonomous mechanosensitivity and serves as a main sensitive receptor in the organ. Piezo1, together with TREK-1 channel, could play a role of local mechanoreceptors in bladder detrusor smooth muscle (DSM) cells. Piezo1 is a so-called “true” mechanosensitive calcium-permeable ion channel, sensitive to pressure, shear stress, and is activated by pharmacological agonist Yoda1. Using patch-clamp and microfluorescence calcimetry, an unexpected effect from Yoda1 was shown, that is inhibition of rest K+ currents at depolarizing command voltage up to +80 mV. The functional presence of Piezo1 is confirmed by Yoda1-induced rise of intracellular Ca2+ concentration in DSM cells, this was visualized using a Ca2+ sensitive dye Fluo-4 AM, and polymerase cyclic reaction with reverse transcription. In conclusion, Piezo1 channels present in DSM cells and are selectively activated with Yoda1, that causes the inhibition of resting potassium currents.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Expression of Calretinin in the Cecal Muscularis Interna: Observation and Hypothetical Relevance to Appendicitis.

The unexpected observation of calretinin immunoreactivity in smooth muscle cells in the muscularis propria of the cecum led to a more detailed examination of calretinin expression and its possible relationship to propulsive contractile activity around the vermiform appendix. Immunohistochemistry and RNA in situ hybridization were performed to analyze calretinin expression in intestinal samples from 33 patients at ages ranging from mid-gestation fetuses to adults, as well as in some potentially relevant animal models. Dual immunolabeling was done to compare calretinin localization with markers of smooth muscle and interstitial cells of Cajal. Calretinin expression was observed consistently in the innermost smooth muscle layers of the muscularis interna in the human cecum, appendiceal base, and proximal ascending colon, but not elsewhere in the intestinal tract. Calretinin-positive smooth muscle cells did not co-express markers located in adjacent interstitial cells of Cajal. Muscular calretinin immunoreactivity was not detected in the ceca of mice or macaques, species which lack appendices, nor in the rabbit cecum or appendix. Localized expression of calretinin in cecal smooth muscle cells may reduce the likelihood of retrograde, calcium-mediated propulsive contractions from the proximal colon and suppress pro-inflammatory fecal stasis in the appendix.

A case of young male with recurrent acute pancreatitis caused by an intrapancreatic gastric duplication cyst

Gastric duplication cyst (GDC) is a rare gastrointestinal malformation that frequently occurs in the greater curvature of the gastric antrum or corpus. Herein, we reported a case of intrapancreatic GDC found as a result of recurring pancreatitis. A 15-year-old man experienced repeated episodes of acute pancreatitis and was found to have a cystic lesion in the pancreatic tail. Contrast-enhanced computed tomography revealed a 20-mm cystic lesion with an enhanced thick wall. Endoscopic ultrasonography revealed an anechoic cyst with a three-layered wall. Magnetic resonance cholangiopancreatography and endoscopic retrograde pancreatography (ERP) revealed a connection between the cyst and the main pancreatic duct (MPD), and the duplication of the MPD. ERP showed the pancreatic duct stenosis downstream of the cyst. Although preoperative diagnosis was difficult, distal pancreatectomy was performed to prevent recurrence of pancreatitis. Pathological examination revealed that the cystic lesion was circumferentially surrounded by the pancreatic parenchyma. The epithelial lining of the cyst was crypt epithelium containing the fundic or pyloric glands and surrounded by a smooth muscle layer. The final diagnosis was intrapancreatic GDC.

3D printed grafts with gradient structures for organized vascular regeneration

Synthetic vascular grafts suitable for small-diameter arteries (<6 mm) are in great need. However, there are still no commercially available small-diameter vascular grafts (SDVGs) in clinical practice due to thrombosis and stenosis after in vivo implantation. When designing SDVGs, many studies emphasized reendothelization but ignored the importance of reconstruction of the smooth muscle layer (SML). To facilitate rapid SML regeneration, a high-resolution 3D printing method was used to create a novel bilayer SDVG with structures and mechanical properties mimicking natural arteries. Bioinspired by the collagen alignment of SML, the inner layer of the grafts had larger pore sizes and high porosity to accelerate the infiltration of cells and their circumferential alignment, which could facilitate SML reconstruction for compliance restoration and spontaneous endothelialization. The outer layer was designed to induce fibroblast recruitment by low porosity and minor pore size and provide SDVG with sufficient mechanical strength. One month after implantation, the arteries regenerated by 3D-printed grafts exhibited better pulsatility than electrospun grafts, with a compliance (8.9%) approaching that of natural arteries (11.36%) and significantly higher than that of electrospun ones (1.9%). The 3D-printed vascular demonstrated a three-layer structure more closely resembling natural arteries while electrospun grafts showed incomplete endothelium and immature SML. Our study shows the importance of SML reconstruction during vascular graft regeneration and provides an effective strategy to reconstruct blood vessels through 3D-printed structures rapidly.

Monotropein Alleviates Ovalbumin-Induced Asthma in Mouse Model by Inhibiting AKT/NF-κB Pathway

Introduction: Clinical management of asthma remains as a prevalent challenge. Monotropein (MON) is a naturally occurring cyclic enol ether terpene glycoside with medical application potential. This study aims to evaluate the potential therapeutic effects of MON in the mouse model of chronic asthma. Methods: An ovalbumin (OVA)-induced asthmatic mouse model was established to evaluate the therapeutic effect of MON at different doses (20, 40, and 80 mg/kg). The potential involvement of protein kinase B (AKT)/nuclear factor kappa B (NF-κB) pathway in the effect of MON was investigated by the administration of an AKT activator SC79. Histological changes in pulmonary tissues were examined by hematoxylin and eosin staining. The profiles of inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13, and tumor necrosis factor [TNF]-α) in bronchoalveolar lavage fluid (BALF), and OVA-specific IgE in blood samples were analyzed by enzyme-linked immunosorbent assay (ELISA). The oxidative stress in the lung tissues was determined by measuring malondialdehyde level. The phosphorylation activation of AKT and NF-κB was examined by immunoblotting in the lung tissues. Results: MON treatment suppressed the infiltration of inflammatory cells in the airways of OVA-induced asthma mice and reduced the thickness of the bronchial wall and smooth muscle layer in a dose-dependent manner. MON treatment also reduced the levels of OVA-specific IgE in serum and cytokines in BALF in asthma-induced mice, and attenuated the oxidative stress in the lung tissues. OVA induced the phosphorylation of AKT and NF-κB proteins in the lung tissues of asthmatic mice, which was significantly suppressed by MON treatment. The co-administration of AKT activator SC79 impaired the therapeutic effect of MON on asthma-induced mice. Conclusion: Our data demonstrated the potential therapeutic effect of MON on asthmatic mouse model, suggesting that MON attenuated the inflammatory and oxidative damages in ling tissues by dampening the AKT/NF-κB signaling pathway.

Inducing aortic aneurysm/dissection in zebrafish: evaluating the efficacy of β-Aminopropionic Nitrile as a model

ABSTRACT Aortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized β-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p < 0.0004, n = 10) and aortic arch malformations, with a high malformation rate of 75% (6/8). Conversely, adult zebrafish showed a 41.67% (5/12) mortality rate 22 days post-induction. At this time point, the dorsal aortic area had expanded by 2.46 times (p < 0.009), and the vessel wall demonstrated significant thickening (8.22 ± 2.23 μM vs. 26.38 ± 10.74 μM, p < 0.05). Pathological analysis revealed disruptions in the smooth muscle layer, contributing to a 58.33% aneurysm rate. Moreover, the expression levels of acta2, tagln, cnn1a, and cnn1b were decreased, indicating a weakened contractile phenotype. Transcriptome sequencing showed a significant overlap between the molecular features of zebrafish tissues post-BAPN treatment and those of AAD patients. Our findings present a straightforward and practical method for generating AAD models in both larval and adult zebrafish using BAPN.

Patient-specific tissue engineered vascular graft for aortic arch reconstruction

Objective(s)The complexity of aortic arch reconstruction due to diverse 3-dimensional geometrical abnormalities is a major challenge. This study introduces 3-dimensional printed tissue-engineered vascular grafts, which can fit patient-specific dimensions, optimize hemodynamics, exhibit antithrombotic and anti-infective properties, and accommodate growth. MethodsWe procured cardiac magnetic resonance imaging with 4-dimensional flow for native porcine anatomy (n = 10), from which we designed tissue-engineered vascular grafts for the distal aortic arch, 4 weeks before surgery. An optimal shape of the curved vascular graft was designed using computer-aided design informed by computational fluid dynamics analysis. Grafts were manufactured and implanted into the distal aortic arch of porcine models, and postoperative cardiac magnetic resonance imaging data were collected. Pre- and postimplant hemodynamic data and histology were analyzed. ResultsPostoperative magnetic resonance imaging of all pigs with 1:1 ratio of polycaprolactone and poly-L-lactide-co-ε-caprolactone demonstrated no specific dilatation or stenosis of the graft, revealing a positive growth trend in the graft area from the day after surgery to 3 months later, with maintaining a similar shape. The peak wall shear stress of the polycaprolactone/poly-L-lactide-co-ε-caprolactone graft portion did not change significantly between the day after surgery and 3 months later. Immunohistochemistry showed endothelization and smooth muscle layer formation without calcification of the polycaprolactone/poly-L-lactide-co-ε-caprolactone graft. ConclusionsOur patient-specific polycaprolactone/poly-L-lactide-co-ε-caprolactone tissue-engineered vascular grafts demonstrated optimal anatomical fit maintaining ideal hemodynamics and neotissue formation in a porcine model. This study provides a proof of concept of patient-specific tissue-engineered vascular grafts for aortic arch reconstruction.

Minimal invasive treatment of urethral strictures: An experimental study of the effect of paclitaxel coated balloons in the wall of strictured rabbit's urethra.

The aim of this study is the evaluation of the distribution of paclitaxel (PTX) released by a coated balloon in the layers of rabbit's urethra. 18 rabbits were included. A laser device was used for the stricture formation. After two weeks, dilation of the strictured urethra was performed by using Advance 35LP PTA balloons and Advance 18 PTX PTA balloons. The experimental models were divided into 3 groups. The group Α included two rabbits without any intervention except for the stenosis procedure. Group B compromised six rabbits that underwent dilation with Advance 35LP PTA balloons. Group C consisted of 10 rabbits to which dilation with both Advance 35LP PTA balloons and Advance 18 PTX PTA balloons was applied. Histological evaluation and Immunohistochemistry were performed on all specimens. Inflammation, fibrosis and ruptures were detected in the specimens of the study. In specimens of Group C the decrease of inflammation and fibrosis rate was greater. Anti-PTX antibody was detected in the epithelium, lamina propria and smooth muscle layer of all specimens of urethras that have been harvested immediately and 1 day after the dilation with Advance 18 PTX PTA balloon and it was not observed in any layer of the urethral wall of the rest of the examined specimens of Group C. PTX's enrichment was detected in the smooth muscle layer of all specimens that have been harvested immediately and 24h after the dilation with Advance 18 PTX PTA balloons. PTX may play an inhibitive role in the recurrence of the stenosis.

Single-cell analysis reveals alterations in cellular composition and cell-cell communication associated with airway inflammation and remodeling in asthma

BackgroundAsthma is a heterogeneous disease characterized by airway inflammation and remodeling, whose pathogenetic complexity was associated with abnormal responses of various cell types in the lung. The specific interactions between immune and stromal cells, crucial for asthma pathogenesis, remain unclear. This study aims to determine the key cell types and their pathological mechanisms in asthma through single-cell RNA sequencing (scRNA-seq).MethodsA 16-week mouse model of house dust mite (HDM) induced asthma (n = 3) and controls (n = 3) were profiled with scRNA-seq. The cellular composition and gene expression profiles were assessed by bioinformatic analyses, including cell enrichment analysis, trajectory analysis, and Gene Set Enrichment Analysis. Cell-cell communication analysis was employed to investigate the ligand-receptor interactions.ResultsThe asthma model results in airway inflammation coupled with airway remodeling and hyperresponsiveness. Single-cell analysis revealed notable changes in cell compositions and heterogeneities associated with airway inflammation and remodeling. GdT17 cells were identified to be a primary cellular source of IL-17, related to inflammatory exacerbation, while a subpopulation of alveolar macrophages exhibited numerous significantly up-regulated genes involved in multiple pathways related to neutrophil activities in asthma. A distinct fibroblast subpopulation, marked by elevated expression levels of numerous contractile genes and their regulators, was observed in increased airway smooth muscle layer by immunofluorescence analysis. Asthmatic stromal-immune cell communication significantly strengthened, particularly involving GdT17 cells, and macrophages interacting with fibroblasts. CXCL12/CXCR4 signaling was remarkedly up-regulated in asthma, predominantly bridging the interaction between fibroblasts and immune cell populations. Fibroblasts and macrophages could jointly interact with various immune cell subpopulations via the CCL8/CCR2 signaling. In particular, fibroblast-macrophage cell circuits played a crucial role in the development of airway inflammation and remodeling through IL1B paracrine signaling.ConclusionsOur study established a mouse model of asthma that recapitulated key pathological features of asthma. ScRNA-seq analysis revealed the cellular landscape, highlighting key pathological cell populations associated with asthma pathogenesis. Cell-cell communication analysis identified the crucial ligand-receptor interactions contributing to airway inflammation and remodeling. Our findings emphasized the significance of cell-cell communication in bridging the possible causality between airway inflammation and remodeling, providing valuable hints for therapeutic strategies for asthma.

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Abstract TMP1: Cerebral Aneurysm Walls Contain Myoglobin That is Possibly Produced by Myofibroblasts and Contributes to Wall Thickening

Background: Cerebral aneurysms rupture at a thin-walled region; however, mechanisms of aneurysmal wall thinning and thickening remain unclear. Myoglobin scavenges nitric oxide and reactive oxygen spices which promote aneurysmal development. Objectives: To investigate the biological role of myoglobin in aneurysmal wall. Methods: Cerebral aneurysmal wall samples were collected between August 2020 and March 2022 for proteomic analysis and immunohistology. We examined the co-localization of myoglobin expression within smooth muscle cells, identified by α-smooth muscle actin (α-SMA) staining, and myofibroblasts, identified by periostin staining. Collagen and myoglobin density in the same sample areas were investigated for its correlation in the aneurysm walls. Results: A total of 35 specimens were obtained from 21 patients who underwent aneurysmal clipping. Proteome analysis was performed using 11 specimens, which firstly detected myoglobin predominantly in the thickened wall. Among 24 formalin-fixed samples, 19 samples were found myoglobin expression. Myoglobin was mostly scattered within the vascular smooth muscle layer and tended to be expressed at sites where iNOS was scarce. Double-label immunofluorescence staining confirmed that the myoglobin-positive rate within α-SMA-positive cells/areas was 33.2±23.8%/31.3±37.8%, respectively, whereas within periostin-positive cells/areas it was 92.2±13.7%/79.8±29.5, respectively. A total of 32 areas were investigated for the correlation between collagen and myoglobin density , which a strong correlation was observed with a Spearman's rank correlation coefficient of 0.635 (p &lt; 0.001). Conclusions: Cerebral aneurysmal walls express myoglobin, which may be produced by myofibroblasts in the wall. Areas with high myoglobin levels retain high levels of collagen fibers, and myoglobin may be involved in wall thickening by suppressing destructive changes in the extracellular collagen fibers.

The conducted vascular response as a mediator of hypercapnic cerebrovascular reactivity: A modelling study

It is well established that the cerebral blood flow (CBF) shows exquisite sensitivity to changes in the arterial blood partial pressure of CO2 (Pa,CO2), which is reflected by an index termed cerebrovascular reactivity. In response to elevations in Pa,CO2 (hypercapnia), the vessels of the cerebral microvasculature dilate, thereby decreasing the vascular resistance and increasing CBF. Due to the challenges of access, scale and complexity encountered when studying the microvasculature, however, the mechanisms behind cerebrovascular reactivity are not fully understood. Experiments have previously established that the cholinergic release of the Acetylcholine (ACh) neurotransmitter in the cortex is a prerequisite for the hypercapnic response. It is also known that ACh functions as an endothelial-dependent agonist, in which the local administration of ACh elicits local hyperpolarization in the vascular wall; this hyperpolarization signal is then propagated upstream the vascular network through the endothelial layer and is coupled to a vasodilatory response in the vascular smooth muscle (VSM) layer in what is known as the conducted vascular response (CVR). Finally, experimental data indicate that the hypercapnic response is more strongly correlated with the CO2 levels in the tissue than in the arterioles. Accordingly, we hypothesize that the CVR, evoked by increases in local tissue CO2 levels and a subsequent local release of ACh, is responsible for the CBF increase observed in response to elevations in Pa,CO2. By constructing physiologically grounded dynamic models of CBF and control in the cerebral vasculature, ones that integrate the available knowledge and experimental data, we build a new model of the series of signalling events and pathways underpinning the hypercapnic response, and use the model to provide compelling evidence that corroborates the aforementioned hypothesis. If the CVR indeed acts as a mediator of the hypercapnic response, the proposed mechanism would provide an important addition to our understanding of the repertoire of metabolic feedback mechanisms possessed by the brain and would motivate further in-vivo investigation. We also model the interaction of the hypercapnic response with dynamic cerebral autoregulation (dCA), the collection of mechanisms that the brain possesses to maintain near constant CBF despite perturbations in pressure, and show how the dCA mechanisms, which otherwise tend to be overlooked when analysing experimental results of cerebrovascular reactivity, could play a significant role in shaping the CBF response to elevations in Pa,CO2. Such in-silico models can be used in tandem with in-vivo experiments to expand our understanding of cerebrovascular diseases, which continue to be among the leading causes of morbidity and mortality in humans.

Implantation of Adipose-Derived Mesenchymal Stromal Cells (ADSCs)-Lining Prosthetic Graft Promotes Vascular Regeneration in Monkeys and Pigs

Background:Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem.Method:We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys.Results:Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome.Conclusion:This cell-based vascular graft is ready to undergo clinical trials for human patients.

Prostatic morphological changes throughout life: Cytochemistry as a tool to reveal tissue aging markers.

The prostate undergoes normal or pathological morphological changes throughout life. An understanding of these changes is fundamental for the comprehension of aging-related pathological processes such as benign prostatic hyperplasia (BPH) and cancer. In the present study, we show some of these morphological changes, as well as histochemical techniques like Weigert's resorcin-fuchsin method, Picrosirius Red, and Gömöri's reticulin for use as tools in the study of prostate tissue under light microscopy. For this purpose, prostates of the Mongolian gerbil (n = 9), an experimental model that develops BPH spontaneously, were analyzed at three life stages: young (1 month old), adult (3 months old), and old (15 months old). The results showed that fibrillar components such as collagen, and reticular and elastic fibers, change throughout life. In young animals, the prostate has cuboidal epithelium surrounded by thin layers of smooth muscle, continuous collagen fibers, winding reticular fibers, and sporadic elastic fibers. With adulthood, the epithelium becomes columnar, encircled by compacted muscle cells among slender collagen fibers, elongated reticular fibers, and linear elastic fibers. In aging individuals, the prostate's epithelium stratifies, surrounded by thick muscle layers among dense collagen fibers, disordered reticular fibers, and elastic fibers in different planes. We also identified a few accumulations of lipid droplets and lipofuscin granules in adult animals and high accumulation in old animals evidenced by Oil red O and Gömöri-Halmi techniques, respectively. The histochemical techniques presented here have been demonstrated to be useful and accessible tools in prostate studies. RESEARCH HIGHLIGHTS: Cytochemical techniques to study prostate morphology. The prostate changes with age.