Expression Of Calponin Research Articles

Pseudo-3D Topological Alignments Regulate Mechanotransduction and Maturation of Smooth Muscle Cells.

Smooth muscle cells (SMCs) sense and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in muscle tissue engineering. Increasing evidence from topological cues-mediated mechanotransduction of SMCs in ECMs has suggested some potential underlying mechanisms of how SMC functions and maturation are regulated by their mechanosensing leading to transduction. However, how the expression of yes-associated protein 1 (YAP) influences the phenotypic shift from synthetic to contractile is still controversial. Here, pseudo-3D topological alignments mimicking native muscle tissues are generated using laser-cutter engraving to explore the influence of topological cues on SMC mechanotransduction and maturation. The analysis of topological cue-mediated mechanotransduction and maturation marker expression revealed YAP is involved in mechanotransduction for SMCs cultured on cross-patterned substrates in the presence of cell-cell interactions. Moreover, these SMCs with YAP-linked mechanosensing showed higher expression of calponin, indicating a shift toward contractile phenotypes in vitro and in vivo. Furthermore, it showed skeletal muscle cells has different mechanosensing and maturation mechanisms compared to SMCs, revealing muscle type-dependent different sensing of topological cues, and converting into maturation-associated signaling cascades. This study provides insights into the regulation of SMC mechanotransduction and maturation by topological cues, suggesting the involvement of YAP-linked signaling pathways in this process.

The modulation of lung cancer cell motility by Calponin 3 is achieved through its ability to sense and respond to changes in substrate stiffness.

The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties.

Research Note: Chicken breast muscle satellite cell function: effect of expression of CNN1 and PHRF1

The Wooden Breast myopathy results in the necrosis and fibrosis of breast muscle fibers in fast-growing heavy weight meat-type broiler chickens. Myogenic satellite cells are required to repair and regenerate the damaged muscle fibers. Using Genome Wide Association, candidate genes affected with Wooden Breast have been previously reported. The effect of these genes on satellite cell proliferation, differentiation, and the synthesis of lipids by satellite cells is unknown. Satellite cells isolated from the pectoralis major muscle from commercial Ross 708 broilers and a Randombred chicken (RBch) line were used. Expression of calponin 1 (CNN1) and PHD and ring fingers domains 1 (PHRF1) were knocked down by silent interfering RNA to determine their effect on satellite cell-mediated proliferation, differentiation, and lipid accumulation. CNN1 and PHRF1 affected satellite cell activity and lipid accumulation in both lines. Proliferation was reduced in the Ross 708 and RBch lines by knocking down the expression of both genes, and differentiation was affected with a line and treatment interaction when gene expression was reduced at the beginning of proliferation. During differentiation lipid accumulation was decreased with knocking down the expression of CNN1 and PHRF1. Both CNN1 and PHRF1 have not been reported previously in skeletal muscle and further research is required to determine their effect on satellite cell-mediated growth and regeneration of the pectoralis major (breast) muscle.

Immunohistochemical marker profiles for the differentiation of collagenous spherulosis from adenoid cystic carcinoma of the breast

Collagenous spherulosis (CS) is a rare breast lesion of unknown histogenesis. Adenoid cystic carcinoma (ACC) is a rare basal-like breast carcinoma with low histological grade. CS is a benign lesion but resembles ACC. Both lesions show a similar histomorphology and feature bilineage differentiation. This study compared immunohistochemical markers in CS and ACC.We compiled n = 13 CS cases and n = 18 mammary ACCs. Fourteen marker proteins (ER, PR, HER2, GATA3, CK7, E-cadherin, CD117, CK5/14, p40, p63, SMA, CD10, calponin, P-cadherin) were evaluated by immunohistochemistry (IHC). MYB rearrangement, a common alteration in ACC, was assessed by fluorescence in situ hybridization.Patient age ranged between 40-60 years for CS lesions and 30–90 years for ACCs. 7/13 (54%) CS cases harbored a lobular carcinoma in situ (LCIS) in the luminal component. One CS/LCIS lesion occurred in a carrier of a pathogenic germline variant in CDH1/E-cadherin. MYB rearrangement was detected in 0/11 (0%) CS and 6/16 (37%) ACC cases (P = 0.054). CS was associated with expression of ER in the luminal component (P < 0.001), E-cadherin loss in the luminal component (P = 0.045), and expression of CD10 and calponin in the basal component (P < 0.001). Furthermore, CS was associated with GATA3 expression in the luminal component (12/13 [92%] versus 5/18 [27%], P < 0.001).In summary, IHC for GATA3 and E-cadherin may contribute to the differential diagnosis between CS and ACC, although these markers are not exclusively expressed in either lesion. Histologic evaluation has to take into account that CS is frequently colonized by LCIS, requiring thorough correlation of histomorphology and immunohistochemical features.

Growth differentiation factor 15 (GDF15) is elevated in human plaques and promotes endothelial-to-mesenchymal transition (EndMT)

Abstract Introduction and Purpose Growth Differentiation Factor 15 (GDF15) is a member of the TGF-β superfamily and is upregulated under conditions of injury and stress such as hypoxia. GDF15 serum concentrations correlate with inflammation, cardiac fibrosis and unfavorable prognosis in cardiovascular diseases. The functional role of GDF15 in endothelial cells is largely unknown. Here, we investigate a possible role of GDF15 in the transdifferentiation of endothelial cells (EC) into mesenchymal cells (EndMT). Methods and Results GDF15 is expressed at mRNA and protein level in primary human ECs and is secreted as determined by qPCR, single cell sequencing (scRNA-seq) and enzyme-linked immunosorbent assay (mean ± SEM, 743.8±39.3 pg/ml, n=3). Analysis of vascular sections from Ldlr-/- mice fed a high-fat diet using spatial transcriptomics revealed increased GDF15 expression. ScRNA-seq analysis of human plaques and normal vessel sections from the same individuals revealed a 22-fold ± 5.48 (p&amp;lt;0.05) increase in GDF15 in ECs located in plaques compared to ECs in control vessel regions. Experiments in cultured human endothelial cells showed increased GDF15 mRNA expression qPCR (+1.7-fold control, p&amp;lt;0.05) and scRNA-seq analysis after EndMT induction. Treatment with increasing levels of recombinant GDF15 (10, 50 and 150 ng/ml) induced EndMT as measured by expression of the EndMT marker calponin (+3.36, 7.99 and 9.53-fold, all p&amp;lt;0.05). Interestingly, EndMT induction decreased extracellular GDF15 levels (-54.6%, p&amp;lt;0.05), whereas intracellular levels in EC were increased (+1.9-fold control, p&amp;lt;0.05). The use of GDF15 neutralising antibodies (1 µg/ml) reduced soluble GDF15 levels by 97.7% as measured by ELISA (p&amp;lt;0.001). Treatment with neutralising antibodies against GDF15 reduced EndMT induction by 54% as measured by the EndMT marker calponin (p&amp;lt;0.05). Conclusion In conclusion, GDF15 is elevated in mouse and human plaques, and in EndMT. Treatment with GDF15 induces EndMT, whereas inhibition by neutralising antibodies reduces EndMT induction. These data are consistent with a functional role of GDF15 in endothelial dysfunction and atherogenesis.

Importance of smooth muscle p110alpha/AKT/FOXO1 signaling for the development of abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs) and extracellular matrix (ECM) degradation. The catalytic PI 3-kinase isoform p110α signals downstream of growth factor receptors. Mice harboring an SMC-specific p110α deficiency (SM-p110α-/-) are characterized by impaired SMC proliferation and survival. Aim We hypothesized that p110α deficiency in SMCs exacerbates the development and progression of AAA as it promotes SMC loss and thus weakens the aortic wall in healthy and diseased animals. Therefore, we investigated how p110α deficiency in SMCs affects AAA formation, vascular integrity as well as downstream signaling in SMCs. Methods and results Medial thickness of the abdominal aorta in SM-p110α-/- mice was significantly decreased compared to wild-type (WT) littermate controls (29.0±3.1µm vs. 42.5±4.1µm). Morphological characterization of the aortic wall by transmission electron microscopy revealed impaired elastic fibers, detached SMCs and apoptotic cells in SM-p110α-/- mice. SMC differentiation markers including calponin and myosin heavy chain were downregulated in the aortic wall of SM-p110α-/- mice. Western blots demonstrated that p110α deficiency impaired the expression of elastin. Consequently, elastin fibers were characterized by reduced fiber width and length. In addition, growth factor stimulated proliferation of p110α-/- SMCs was significantly impaired compared to WT controls. AAA development in SM-p110α-/- mice and WT littermates were studied using the porcine pancreatic elastase (PPE) model. PPE was infused into the infrarenal aorta. Ultrasound examination of the aorta revealed an increased aortic diameter in all PPE-treated mice. However, the increase in aortic diameter was significantly elevated (p&amp;lt;0.05) in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10). Furthermore, PPE treated SM-p110-/- mice were characterized by increased ECM degradation, decreased vascular remodelling, and less proliferating cells. Mechanistically, growth factor-dependent phosphorylation and inactivation of crucial phenotypic modulators of SMCs - FOXO-1, -3 and -4 - were shown to be significantly decreased in p110α-/- SMCs compared to WT cells. Specific inhibition of FOXO1 rescued the impaired proliferation of p110α-/- SMCs and enhanced the expression of elastin and calponin. Analysis of FOXO expression in humans revealed that FOXO1 was significantly upregulated in AAA patients compared to healthy individuals. Conclusion p110α deficiency in SMCs promotes the development and progression of AAA, as p110α controls crucial SMC functions which are important for structural processes in the aortic wall. Thereby, SMC proliferation is controlled by p110α dependent phosphorylation and inactivation of FOXO1. Since FOXO1 is upregulated in AAA patients, the p110α/AKT/FOXO1 signaling cascade may provide a key target for modulating aortic wall stability in AAA.

Heterotopia of salivary gland tissue in the pancreas

Heterotopia of the salivary gland occurs mainly in the head and neck region of the human body, rarely in regions such as the rectum, but has never been demonstrated in the pancreas. Within a screening effort of pancreatic samples for detecting ΔNp63 expression, we discovered two pancreatic samples from a 35-year-old male showing salivary gland heterotopia. Immunohistochemical stainings were done for markers of healthy and neoplastic salivary glands and showed expression of calponin, CD142 and KRT14 but not of S100p, GFAP or CD117. A PAS-staining and Alcian Blue staining showed the presence of acid mucins. These staining patterns were consistent with non-neoplastic submandibular gland tissue comprised of abundant seromucous glands, basal cells and myoepithelial cells, all features typically absent in the pancreas. Also, no pancreatic islets of Langerhans were detected. We show for the first time that salivary gland heterotopia can occur at the location of the pancreas.

Human aortic smooth muscle cell regulation by METTL3 via upregulation of m6A NOTCH1 modification and inhibition of NOTCH1.

Thoracic aortic dissection (TAD) is a very serious vascular condition that requires immediate treatment. Phenotypic conversion of human aortic smooth muscle cells (HASMCs) has been reported to be a causal factor for TAD development. Genetic variations affecting RNA modification may play a functional role in TAD. In this study, we aimed to explore the potential role of the methyltransferase like 3 (METTL3) and notch homolog 1 (NOTCH1) N6-methyladenosine (m6A) modification mechanisms in HASMCs. HASMCs were cultured. METTL3 was knocked down and overexpressed. Then, both METTL3 and NOTCH1 were simultaneously knocked down in HASMCs. HASMC proliferation was determined using Cell Counting Kit-8(CCK-8). METTL3, NOTCH1, α-smooth muscle actin (α-SMA), smooth muscle protein 22-alpha (SM22α), and calponin expressions were monitored with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An m6A dot blot assay was used to examine the m6A modification levels. The NOTCH1 3' untranslated region (3'UTR) m6A modification was analyzed using SRAMP and RMBase v. 2.0. A methylated RNA immunoprecipitation (MeRIP) assay was used to evaluate the METTL3 overexpression effect on m6A modification of NOTCH1 messenger RNA (mRNA). A dual-luciferase assay was used to investigate the effect of METTL3 binding of the NOTCH1 mRNA m6A modification site. YTH domain family 2 (YTHDF2)-RNA immunoprecipitation (RIP) was used to detect the change in YTHDF2's ability to bind to NOTCH1 mRNA after METTL3 overexpression. Overexpression of METTL3 inhibited α-SMA, SM22α, calponin, and NOTCH1 expressions and promoted HASMC proliferation. Knocking down METTL3 had the opposite effect. The cointerference of the METTL3 and NOTCH1 results suggested that METTL3 regulated NOTCH1, contributing to HASMC phenotypic changes. The MeRIP assay showed that the m6A modification of NOTCH1 mRNA increased after METTL3 overexpression. The dual-luciferase assay indicated that the NOTCH1 mRNA m6A modification site and METTL3 overexpression promoted NOTCH1 mRNA degradation. YTHDF2-RIP further demonstrated that the binding ability of YTHDF2 and NOTCH1 mRNA was enhanced after METTL3 overexpression. METTL3 regulated the phenotypic changes of HASMC by upregulating m6A modification of NOTCH1 and inhibiting NOTCH1.

Flexible, Suturable, and Leak-free Scaffolds for Vascular Tissue Engineering Using Melt Spinning.

Coronary artery disease affects millions worldwide. Bypass surgery remains the gold standard; however, autologous tissue is not always available. Hence, the need for an off-the-shelf graft to treat these patients remains extremely high. Using melt spinning, we describe here the fabrication of tubular scaffolds composed of microfibers aligned in the circumferential orientation mimicking the organized extracellular matrix in the tunica media of arteries. By variation of the translational extruder speed, the angle between fibers ranged from 0 to ∼30°. Scaffolds with the highest angle showed the best performance in a three-point bending test. These constructs could be bent up to 160% strain without kinking or breakage. Furthermore, when liquid was passed through the scaffolds, no leakage was observed. Suturing of native arteries was successful. Mesenchymal stromal cells were seeded on the scaffolds and differentiated into vascular smooth muscle-like cells (vSMCs) by reduction of serum and addition of transforming growth factor beta 1 and ascorbic acid. The scaffolds with a higher angle between fibers showed increased expression of vSMC markers alpha smooth muscle actin, calponin, and smooth muscle protein 22-alpha, whereas a decrease in collagen 1 expression was observed, indicating a positive contractile phenotype. Endothelial cells were seeded on the repopulated scaffolds and formed a tightly packed monolayer on the luminal side. Our study shows a one-step fabrication for ECM-mimicking scaffolds with good handleability, leak-free property, and suturability; the excellent biocompatibility allowed the growth of a bilayered construct. Future work will explore the possibility of using these scaffolds as vascular conduits in in vivo settings.

Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion.

Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells, and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vascular ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model. HUVECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-β1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM), and HUVEC+DPSC cocultures (E+D-CM) were analyzed by enzyme-linked immunosorbent assay. TGF-β RI kinase inhibitor VI, SB431542, was used to block TGF-β1/ALK5 signaling in DPSCs. The expression of SMC-specific markers, α-SMA, SM22α, and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-β1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-β1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-β1/ALK5 signaling inhibited the expression of α-SMA, SM22α, and Calponin in DPSCs. TGF-β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-β1/ALK5 signaling pathway played a vital role in this process.

#3986 THE ROLE AND MECHANISM OF EZH2 IN VASCULAR CALCIFICATION ASSOCIATED WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Vascular calcification (VC) is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. EZH2 (Enhancer of zeste 2 homolog-2) is reported as a key epigenetic regulator involved in various kidney diseases such as acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. In this study, we aimed to investigated the role of EZH2 in chronic kidney disease associated vascular calcification. Method 1. Patients and Radial Artery Analysis Radial arteries with or without calcification from patients with end-stage renal disease that underwent arterial venous fistular surgery were collected for Von Kossa staining and immunohistochemical (IHC) staining. 2. Animal model of Chronic renal failure-Vascular Calcification (CRF-VC) (in vivo) An adenine and phosphate (1.2%) diet-induced CRF mouse model was designed following an 8-week program. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition and histological analysis. Plasma levels of Alkaline phosphate (ALP), and phosphate (Pi) were measured. The transcription level of EZH2 and Runt-related transcription factor 2 (Runx2) were measured by real-time PCR and IHC staining. 3. Aortic ring calcification (ex vivo) Thoracic aortas were dissected from SD rat. To induce calcification, Pi (NaH2PO4/Na2HPO4 [pH = 7.4]) was added to HG-DMEM at a final concentration of 2.6 mmol/l. After the indicated incubation periods (3, 5, 7 days), samples were taken for Calcium deposition assay, Western blotting and histological analysis. 4. Cell culture and treatment (in vitro) Calcification of rat smooth muscle embryonic thoracic aorta cell line A7r5 (VSMC) was induced by incubation in calcifying media. 3-deazaneplanocin A (3-DZNeP), a carbocyclic analog of adenosine was added right after Pi to block EZH2 activation. After the indicated incubation periods, samples were collected for Calcium deposition assay and Western blotting analysis. Results 1. In vivo, EZH2 protein level was increased in calcified radial arteries from patients with end-stage renal disease that underwent arterial venous fistular operation. In CRF-VC mice model, the aortic calcium deposition markedly aggravated in the group induced by adenine diet. Severe vascular calcification was also induced as shown by increased intensity of von Kossa. Moreover, IHC and q-PCR results showed that the protein and transcriptional levels of EZH2 expression were upregulated in calcified aortas of CRF mice, in paralleled with the increased expression Runx2 in protein and transcriptional levels. 2. Ex vivo, histological analysis revealed significant medial vascular calcification in the aortic ring culture with this calcifying medium for 7 days. Similarly, WB results showed that the expression of EZH2 and its downstream, methylation of Histone H3 at lysine 27 (H3K27me3), were significantly enhanced after high Pi medium stimulation while the osteogenic markers Runx2, Osteopontin (OPN) and Msh homeobox 2 (Msx2) protein levels were upregulated and the expression of VSMC differentiation markers α-Smooth muscle actin (α-SMA), Calponin (CNN) and Smooth muscle protein 22 (SM22) were downregulated. 3. In vitro, the osteogenic markers Runx2, OPN and Msx2 were significantly upregulated in high Pi treated VSMCs compared to control VSMCs. Moreover, the epigenetic markers EZH2 and H3K27me3a were markedly repressed in calcifying VSMCs by time. 4. 3-DZNep treatment reduced the calcium deposition in a concentration dependent manner. Western blotting showed that 3-DZNep treatment blocked the increased expression of osteogenic markers Runx2, Bone morphogenetic protein 2 (BMP2) as well as epigenetic markers EZH2 and H3K27me3a by high Pi treatment, indicating that inhibition of EZH2 attenuates VSMCs Calcification. Conclusion Our study revealed that EZH2 is highly expressed in calcified vascular tissues of CRF patients and CRF mice, rat aorta culture and VSMCs calcification models, and EZH2 inhibitor 3-DZNep attenuated calcification of VSMCs. EZH2 could be a promising target for treatment of vascular calcification in CRF patients.

ROLE OF EXPRESSION OF P63 AND CALPONIN IN PROSTATIC BIOPSIES

Background: An increased number of mortality and morbidity in elderly men is caused due to prostatic diseases all over the world. An increase in mass screening programmes conducted with the help of Prostate Specic Antigen (PSA) blood test along with Digital rectal examination (DRE) and imaging studies has resulted in an upsurge in the diagnosis of prostatic cancer which is markedly noted in the recent decades. Material And Methods: A total 100 cases of formalin xed parafn embedded histological sections of various prostatic lesions including benign prostatic hyperplasia, prostatic intraepithelial lesion and prostate carcinoma were studied. Out of 100 cases, 55 cases were benign prostatic hyperplasia, 10 cases of prostatic intraepithelial lesion and 35 cases were of prostate adenocarcinoma. All the sections were stained with Hematoxylin and Eosin stain and then immunohistochemical staining of p63 and Calponin was performed on all the cases (gure 6- 12). The parameters used to analyse the expression of both p63 and calponin were - Pattern of staining, Percentage of stained cell, Intensity of staining reaction. Total 100 cases i Results: ncluded in this study, out of which 55 (55%) cases were benign prostatic hyperplasia, 10 (10%) cases of prostatic intraepithelial neoplasia, and 35 (35%) cases were prostatic adenocarcinoma, majority of specimens (78%) were TURP followed by trucut biopsy (20%), Suprapubic prostatectomy (2%). The proportion of Transurethral resection of prostate (TURP) was signicantly higher as compared to the proportion of Trucut biopsy and supra pubic prostatectomy (SPP) (p&lt;.001). Expression of calponin in various prostatic lesion. On analysing expression of proliferation marker calponin, in BPH it was expressed in only 52 cases out of 5 cases (94.5%) and 3 cases (8.6%) were negative. Our study concluded that the proportion of Benign prostatic Conclusion: hyperplasia was signicantly higher as compared to the Prostatic intraepithelial neoplasia and Prostatic adenocarcinoma. Immunohistochemical staining by p63 is diagnostically reliable in identifying basal cells in prostatic needle biopsies and TURP specimens. p63 is a valuable tool with high sensitivity in differentiating BPH from prostatic carcinoma. The decreased or absent stromal staining of calponin which indicates a reactive stroma in malignancy can be used alone or in conjunction with p63 for conrmation of diagnosis of prostatic carcinoma

Mechanism of Notch3 signaling pathway regulating the differentiation of aortic dissection vascular stem cells into smooth muscle cells

To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process. Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting. Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05). Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.

Short Term Effect of Gamma-Irradiation on Cytokeratin 17 and Calponin Expression in Submandibular Glands of Rats

Short Term Effect of Gamma-Irradiation on Cytokeratin 17 and Calponin Expression in Submandibular Glands of Rats

Retinol binding protein 4 promotes the phenotypic transformation of vascular smooth muscle cells under high glucose condition via modulating RhoA/ROCK1 pathway

Phenotypic switch of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS). High level of retinol binding protein 4 (RBP4) is regarded as a risk factor in cardiac-cerebral vascular disease. This study is performed to clarify the biological function of RBP4 in modulating the phenotypic switch of VSMCs induced via RhoA/ROCK1 signaling pathway. In vivo experiment, all the rats were divided into control group (NC), diabetic group (DM) and diabetic atherosclerosis group (DAS). The expressions of biochemical indicators, RhoA and Rho associated coiled-coil containing protein kinase 1 (ROCK1) were detected. In vitro experiment, VSMCs were cultured under high glucose condition, and ectogenic RBP4, HA-1100, rapamycin, or 3-methyladenine (3-MA) were supplemented to treat the VSMCs, respectively. The proliferation and migration of VSMCs were evaluated. The regulatory relationship between RBP4 and ROCK1 was predicted by bioinformatics analysis, and validated by qRT-PCR and Western blot. The regulatory effects of RBP4 on contractile phenotypic markers such as calponin, MYH11, α-SMA and autophagy markers including LC3II, LC3I, and Beclin-1 as well as mTOR were also detected. Moreover, VSMCs were cultured exposed to ROCK1 overexpressed plasmid or short hairpin RNA (shRNA), the proliferation and migration of VSMCs were evaluated and the regulatory effects of RhoA/ROCK1 signaling pathway on contractile phenotypic markers and autophagy markers were also detected. In vivo, RhoA, ROCK1, and mTOR were highly expressed in the rats intraperitoneally injected with RBP4. In vitro, the expressions of calponin, MYH11, α-SMA, LC3II, LC3I, and Beclin-1 were decreased in VSMCs treated with ROCK1-OA under high glucose condition, conversely, the expressions were increased in VSMCs exposed to ROCK1-shRNA. After incubated with rapamycin additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were up-regulated and the expression of p-mTOR was decreaed in VSMCs of HG+ROCK1-OA. Conversely, after incubated with 3-MA additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were down-regulated and the expression of p-mTOR was elevated in VSMCs of HG+ROCK1-shRNA. Ectogenic RBP4 facilitated high glucose-induced proliferation and migration of VSMCs, and it repressed the expression of calponin, MYH11, α-SMA, LC3II/I, and Beclin-1 in VSMCs. As expected, ROCK1 inhibit or counteracted the biological effects of RBP4 on VSMCs. In addition, the expressions of contractile phenotypic markers, LC3II/I, and Beclin-1 were promoted and mTOR were decreased after the VSMCs treated with autophagy agonist, whereas no significant difference was observed in the expressions of ROCK1, RhoA. RBP4 is an injurious factor in the pathogenesis of diabetic AS, and it promotes the phenotypic switch of VSMCs via activating RhoA/ROCK1 pathway and inhibiting autophagy.

Trehalose attenuates abdominal aortic aneurysm formation by inducing autophagy in smooth muscle cells.

Trehalose is a naturally occurring disaccharide, which has been identified as an autophagy inducer and exhibits protective effect in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of trehalose in abdominal aortic aneurysm (AAA) remains undefined. To study the effect of trehalose in AAA, trehalose (1 g/kg per day) were given for 14 continuous days in a mouse model of elastase-induced abdominal aortic aneurysm. On day 14, ultrasound was performed to measure aortic diameter before the abdominal aortas were harvested and processed for further analysis. Verhoeff-Van Gieson staining and TUNEL staining were performed on paraffin sections to evaluate vascular histology and apoptosis, immunofluorescence staining and Western-blot were performed to evaluate expression of autophagy markers. Echocardiography and in situ pictures demonstrated that trehalose attenuated infrarenal aorta dilation. Verhoeff-Van Gieson staining showed elastin degradation was improved in trehalose-treated group. Compared with vehicle-treated mice, trehalose treatment restored smooth muscle cell contractile phenotype with increased α-SMA, Calponin and Myh11 expression. Furthermore, trehalose also attenuated cell apoptosis and leukocytes infiltration. Importantly, trehalose induced autophagy with decrease SQSTM1/p62 accumulation, increased lamp2 expression and LC3B conversion. Trehalose attenuated AAA progression with decreased inflammation and restored SMC contractile phenotype by inducing autophagy. These results demonstrated the therapeutic potential of trehalose in AAA.

Experimental conditions and protein markers for redifferentiation of human coronary artery smooth muscle cells.

A phenotype switch from contractile type to proliferative type of arterial smooth muscle cells is known as dedifferentiation, but to the best of our knowledge, little is known about redifferentiation of coronary artery smooth muscle cells. The purpose of the present study was to determine in vitro culture conditions for inducing redifferentiation of coronary artery smooth muscle cells. In addition, the present study aimed to determine protein markers for detection of redifferentiated arterial smooth muscle cells. Human coronary artery smooth muscle cells (HCASMCs) were cultured in the presence or absence of growth factors, including epidermal growth factor, fibroblast growth factor-B and insulin. Protein expression and migration activity of HCASMCs were evaluated using western blotting and migration assay, respectively. In HCASMCs 5 days after 100% confluency, expression levels of α-smooth muscle actin (α-SMA), calponin, caldesmon and SM22α were significantly increased, while expression levels of proliferation cell nuclear antigen (PCNA) and S100A4 and migration activity were significantly decreased, compared with the corresponding levels just after reaching 100% confluency, indicating that redifferentiation occurred. Redifferentiation was also induced in a low-density culture of HCASMCs in the medium without growth factors. When the culture medium for confluent cells was replaced daily with fresh medium, the expression levels of α-SMA, caldesmon, SM22α, PCNA and S100A4 and migration activity were not significantly different but the calponin expression was significantly increased compared with the levels in dedifferentiated cells just after reaching 100% confluency. Thus, redifferentiation was induced in HCASMCs by deprivation of growth factors from culture medium. The results suggested that α-SMA, caldesmon and SM22α, but not calponin, are markers of redifferentiation of HCASMCs.

Breast myofibroblastoma in a woman: a case report

Breast myofibroblastoma is a rare benign neoplasm of mesenchymal origin with fibroblastic and myofibroblastic characterizations. Myofibroblastoma has a higher incidence in men between the ages of 50 and 70 years and is less common in women. It is described as a solitary, unilateral, painless and mobile tumor, with a firm consistency and slow growth. Microscopically, it is a non-encapsulated tumor, with lobular growth, consisting of spindle cells organized in short, intersecting fascicles and interrupted by bundles of hyalinized collagen. On ultrasound, it can manifest as a hypoechoic solid mass, well circumscribed, homogeneous and similar to fibroadenoma; whereas, on mammography, a single, well-defined, rounded or discretely lobulated lesion can be observed, without calcifications. We report here the case of a 58-year-old patient with no previous breast complaints, who presented with changes in ultrasound and mammography examinations performed for breast cancer screening. The examinations revealed a suspicious lump in the left breast, classified as BIRADS 4C. Core biopsy described a low-grade spindle cell neoplasm, showing no signs of invasion, with immunohistochemistry results suggesting myofibroblastoma. As treatment, a sectorectomy was performed, and the reevaluated material confirmed the diagnosis of myofibroblastoma due to the positive expression of the markers calponin, CD34, BCL2 and CD99.

Calponin 1 increases cancer-associated fibroblasts-mediated matrix stiffness to promote chemoresistance in gastric cancer

The mechanical microenvironment regulated by cancer-associated fibroblasts (CAFs) influence tumor progression. Chemotherapeutic interventions including 5-Fluorouracil (5-Fu) are commonly used for primary treatment of patients with advanced gastric cancer (GC), and the development of acquired resistance to 5-Fu limits the clinical efficacy of these chemotherapies. However, if and how the interplay between CAFs and the mechanical microenvironment regulates GC response to 5-Fu is poorly understood. In this study, we demonstrate that high-level expression of calponin 1(CNN1) in gastric CAFs predicts poor clinical outcomes of GC patients, especially for those treated with 5-Fu. CNN1 knockdown in CAFs improves the effectiveness of 5-Fu in reducing tumor growth in a mouse GC model and confers increased sensitivity to 5-Fu in a 3D culture system. Furthermore, CNN1 knockdown impairs CAF contraction and reduces matrix stiffness without affecting the expression of matrix proteins. Mechanistically, CNN1 interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, which leads to activation of the ROCK1/MLC pathway. The increased matrix stiffness, in turn, contributes to 5-Fu resistance in GC cells by activating YAP. Taken together, our data reveal a critical role of the mechanical microenvironment in 5-Fu resistance, which is modulated by CNN1hi CAFs-mediated matrix stiffening, indicating that targeting CAFs may provide a novel option for overcoming drug resistance in GC.

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P&amp;lt;0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P&amp;lt;0.05) and VCAN (2.0-fold, P&amp;lt;0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P&amp;lt;0.05) and Calponin (−93.8%, P&amp;lt;0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P&amp;lt;0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)