Interstitial Cell Research Articles

Granulomatous lobular mastitis co-existing with ductal carcinoma in situ: Report of three cases and review of the literature

Granulomatous lobular mastitis (GLM) is a benign and infrequent chronic breast ailment. Although this lesion can be clinically and radiographically mistaken for early-onset breast cancer, it is a rare occurrence for the two to coexist. This report describes three such cases. In all three patients, the primary signs and symptoms were related to the formation of diffuse breast masses or abscesses. Breast ultrasound and MRI revealed glandular edema and dilated breast ducts. The biopsies of all lesions exhibited both granulomatous inflammation confined to the lobules of the breast, abundant interstitial inflammatory cell infiltrates, and apparently cancerous cells located in dilated ducts with intact basement membranes. The surgically excised specimens confirmed the diagnosis of GLM and ductal carcinoma in situ (DCIS) in all three patients who underwent breast mass resection. By clinical imaging and clinical manifestations, GLM may obscure a concurrent DCIS, as highlighted by the cases reported herein.

Telocytes and inflammation: A review.

Telocytes are a new type of interstitial cell with a diverse morphology and important functions, such as mechanical support, signal transduction, immune regulation, and tissue repair. In this paper, the origin and physiological and pathological functions of telocytes as well as their role in inflammation will be discussed, and the functions and targets of telocytes in inflammation will be fully reviewed, which may contribute to a new therapeutic strategy for inflammatory diseases in the future.

Ultrastructural Architecture Studies of Pancreas in Guinea pig

Background: The pancreas is an accessory organ of the digestive system and also an important endocrine organ of vertebrates that produce and release substances in the body. The pancreas has both endocrine and exocrine function. Its endocrine function is to regulate blood sugar levels by secretion of hormones like insulin, glucagon, stomatostatin and pancreatic polypeptide and an exocrine function that helps in digestion. The study was performed to document the ultrastructural details of pancreas of guinea pigs by scanning and transmission electron microscopy. Methods: Six adult healthy guinea pigs of 16-32 weeks of age (Irrespective of sex) were procured from the Department of Laboratory Animal Medicine, TANUVAS as per ethical committee approval. Animals were dissected according to standard operating procedure by using the Carbon dioxide asphyxiations as per CPCSEA norms and pancreatic pieces were utilised for SEM and TEM study. Result: Pancreas was irregular in shaped and showed splenic, ventricular and intestinal lobes. In SEM, the parenchyma was covered by the dense irregular capsule. Each lobule contained many acini which were connected by a thin, long duct with branched pattern arrangement with increasing wall thickness and diameter. In TEM, the pancreatic tissue consisted of glandular lobules comprised of acini, islets of Langerhans and connective tissue between the lobules. Numerous mitochondria and golgi complexes were also present in the acinar cell cytoplasm along with zymogen granules and rough endoplasmic reticulum. The centroacinar cells were also found. A special type of interstitial cell named telocytes and each was found with many telopodes in the exocrine part of pancreatic parenchyma. Among the four islet cell types, alpha and beta cells could be identified.

Expression and functional study of cholecystokinin-A receptors on the interstitial Cajal-like cells of the guinea pig common bile duct.

Many studies have shown that interstitial Cajal-like cell (ICLC) abnormalities are closely related to a variety of dynamic gastrointestinal disorders. ICLCs are pacemaker cells for gastrointestinal movement and are involved in the transmission of nerve impulses. To elucidate the expression profile and significance of cholecystokinin-A (CCK-A) receptors in ICLCs in the common bile duct (CBD), as well as the role of CCK in regulating CBD motility through CCK-A receptors on CBD ICLCs. The levels of tyrosine kinase receptor (c-kit) and CCK-A receptors in CBD tissues and isolated CBD cells were quantified using the double immunofluorescence labeling technique. The CCK-mediated enhancement of the movement of CBD muscle strips through CBD ICLCs was observed by a muscle strip contraction test. Immunofluorescence showed co-expression of c-kit and CCK-A receptors in the CBD muscularis layer. Observations of isolated CBD cells showed that c-kit was expressed on the surface of ICLCs, the cell body and synapse were colored and polygonal, and some cells presented protrusions and formed networks adjacent to the CBD while others formed filaments at the synaptic terminals of local cells. CCK-A receptors were also expressed on CBD ICLCs. At concentrations ranging from 10-6 mol/L to 10-10 mol/L, CCK promoted CBD smooth muscle contractility in a dose-dependent manner. In contrast, after ICLC removal, the contractility mediated by CCK in CBD smooth muscle decreased. CCK-A receptors are highly expressed on CBD ICLCs, and CCK may regulate CBD motility through the CCK-A receptors on ICLCs.

Recurrence of follicular dendritic cell sarcoma in tonsils after 7 years: a rare case report

Follicular dendritic cell sarcoma (FDCS) is a rare mesenchymal neoplasm. It arises from not only from lymph nodes but also from extra nodal tissues, either as acquired lymphoid tissue or as part of the organized constitutive lymphoid tissue. We here report this rare entity which developed again after 7 years post tonsillectomy in a 5-year old male patient. Patient underwent radical tonsillectomy and adjuvant treatment is awaited. Differential diagnosis includes large cell lymphoma, peripheral nerve sheath tumor, extracranial meningioma, malignant melanoma, metastatic carcinoma, ectopic thymoma, malignant fibrous histiocytoma, and interstitial reticulum cell sarcoma. Currently, the management of FDCS includes the therapeutic guidelines similar to that of high-grade soft tissue sarcomas that is complete surgical resection of the lesion with possibility of adjuvant radiotherapy and/or chemotherapy. The ideal combination of management of FDCS has yet to be defined.

Silk Dye Effluent Induced Change in Ultra-Structure of Testis on Swiss Albino Male Mice Mus Musculus and Recovery by Moringa Oleifera Leaves Powder

The silk dye effluent is one of the most potential harmful chemicals liberated in the environment in an unexpected manner. Silk dye waste is widely used as a potent dyeing of yarn and fabrics in many countries and has been shown to produce some adverse health effects. This work focuses primarily on the effects of Moringa oleifera leaf extract on testis of silk dye effluent induced surface ultrastructure in Swiss albino mice Mus musculus. The testis has been taken an account for surface ultrastructural study. The mice were divided into 5 Groups i.e. Group I (Control), Group II (fed with 50% silk dye), Group III (fed with 100% silk dye), Group IV (mice fed with 50% dye treated with M. oleifera leaves powder), Group V (mice fed with 100% dye treated with M. oleifera leaves powder) have been taken for experiment. The dose of silk dye was 2ml/day to both groups II and III and M. oleifera leaf is given as per the standard dose (300mg/kg b.w) to both animals of group IV and V. Administration of silk dye waste result were demonstrated disrupted spermatids, cessation of seminiferous tubule cell, irregular cavity, lump of seminiferous tubules, disrupted interstitial cell and occupied spermatogonial cells but used of Moringa oleifera leafs powder it was significantly recovered the damage tissues has been observed. This study suggested that the extract may have beneficial effect on surface ultrastructural constituents such as Testis.
 Key words: Silk dye Effluent, Moringa oleifera leaf powder, Testis, Mus musculus, SEM for Ultra-Structure, Toxicity assessment.

Proteomic analysis of acute radiation-induced rectal injury in rats

ObjectiveTo comprehensively elucidate overall protein alterations associated with acute radiation-induced rectal injury in rats. MethodsA rat model of acute radiation-induced rectal injury was established by irradiating rectal segments with a single dose of 17.5 ​Gy X-rays. These segments were then collected at 7 d and 10 ​d post-irradiation. Tandem mass tag-based quantitative proteomic analysis was then performed. Results65,526 peptides were identified, corresponding to 8,088 proteins. Hematoxylin-eosin staining revealed characteristic epithelial cell degeneration and necrosis, intestinal gland atrophy and dilatation, and interstitial inflammatory cell infiltration. Inflammation was more pronounced in the 10 d irradiation group than in the 7 d irradiation group. Overall, 127 up- and 108 downregulated proteins were identified at 7 d post-irradiation, and 122 up- and 44 downregulated proteins were identified at 10 d post-irradiation. Notably, 17 up- and 6 downregulated proteins were consistently co-expressed at both time points. The expression of three of these proteins was validated via real-time quantitative PCR: polypeptide YY (Pyy), thymidylate synthase (Tyms), and tetraspanin (CD9). Tyms transcript levels were significantly higher in irradiated rectal tissues (P ​< ​0.05). Pyy transcript levels were significantly higher at both time points (P ​< ​0.05). Finally, CD9 mRNA expression was significantly lower in both the 7 d and 10 d irradiation groups (P ​< ​0.05). ConclusionsThe potential targets were found to prevent and treat acute radiation-induced rectal injury in clinical practice.

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Rifampicin induced acute interstitial nephritis in a patient with tuberculosis: A case report

Interstitial nephritis is a rare complication of anti-tuberculosis treatment, most commonly rifampicin. This case report describes a 65-year-old woman with diabetes who presented with loss of consciousness due to a major hypoglycemic episode precipitated by severe acute kidney injury, one month after initiating anti-tuberculous therapy. The presence of active urinary sediments, subnephrotic proteinuria and normal sized kidneys prompted performing a renal biopsy which showed interstitial inflammatory cell infiltration suggestive of acute interstitial nephritis, possibly drug induced. The reversal of renal impairment on cessation of rifampicin confirmed the diagnosis. As this is a reversible cause of acute kidney injury, awareness of this association and early identification is vital to prevent deterioration.

Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart

Objective: Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Methods and Results: To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression in vivo . Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis. Conclusions: The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.

Abstract P1178: APOE-NOTCH Axis Governs Elastogenesis During Human Cardiac Valve Remodeling

Background: Valve remodeling is a complex process involving extracellular matrix organization, development of trilaminar structures, and physical elongation of valve leaflets. However, the cellular and molecular mechanisms regulating valve remodeling and their roles in congenital valve disorders remain poorly understood. Methods: Semilunar valves and atrioventricular valves from healthy and age-matched human fetal hearts with pulmonary stenosis (PS) were collected. Single-Cell RNA-sequencing (scRNA-seq) was performed to determine the transcriptomic landscape of multiple valvular cell subtypes in valve remodeling and disease. Results: scRNA-seq analysis of healthy human fetal valves identified a novel APOE+ elastin-producing valve interstitial cell (Elastin-VIC) subtype spatially located underneath VECs in the direction of unidirectional flow. Knockdown of APOE in fetal VICs resulted in significant elastogenesis defects. In pulmonary valve with PS, we observed decreased expression of APOE and other genes regulating elastogenesis such as EMILIN1 and LOXL1 , as well as elastin fragmentation. Cell-cell interaction analysis revealed that JAG1 from unidirectional VECs activates NOTCH signaling in Elastin-VICs through NOTCH3. In vitro Jag1 treatment in VICs increased the expression of elastogenesis-related genes and enhanced contractile functions with related gene expression. This was accompanied by activation of NOTCH signaling and elastogenesis observed in VICs co-cultured with VECs in the presence of unidirectional flow. Notably, we found that the JAG1-NOTCH3 signaling pair was drastically reduced in the PS valves. Lastly, we demonstrated that APOE is indispensable for JAG1-induced NOTCH activation in VICs, reinforcing the presence of a synergistic intrinsic and external regulatory network involving APOE and NOTCH signaling that is responsible for regulating elastogenesis during human valve remodeling. Conclusion: scRNA-seq analysis of human fetal valves identified a novel Elastin-VIC subpopulation, and revealed mechanism of APOE and NOTCH signaling mediated elastogenesis during valve remodeling, which provided opportunity to understand pathogenesis of elastic malformation within congenital valve disease.

Regulatory roles of Osteopontin in lung epithelial inflammation and epithelial-telocyte interaction.

Lung epithelial cells play important roles in lung inflammation and injury, although mechanisms remain unclear. Osteopontin (OPN) has essential roles in epithelial damage and repair and in lung cancer biological behaviours. Telocyte (TC) is a type of interstitial cell that interacts with epithelial cells to alleviate acute inflammation and lung injury. The present studies aim at exploring potential mechanisms by which OPN regulates the epithelial origin lung inflammation and the interaction of epithelial cells with TCs in acute and chronic lung injury. The lung disease specificity of OPN and epithelial inflammation were defined by bioinformatics. We evaluated the regulatory roles of OPN in OPN-knockdown or over-expressed bronchial epithelia (HBEs) challenged with cigarette smoke extracts (CSE) or in animals with genome OPN knockout (gKO) or lung conditional OPN knockout (cKO). Acute lung injury and chronic obstructive pulmonary disease (COPD) were induced by smoking or lipopolysaccharide (LPS). Effects of OPN on PI3K subunits and ERK were assessed using the inhibitors. Spatialization and distribution of OPN, OPN-positive epithelial subtypes, and TCs were defined by spatial transcriptomics. The interaction between HBEs and TCs was assayed by the co-culture system. Levels of OPN expression increased in smokers, smokers with COPD, and smokers with COPD and lung cancer, as compared with healthy nonsmokers. LPS and/or CSE induced over-production of cytokines from HBEs, dependent upon the dysfunction of OPN. The severity of lung inflammation and injury was significantly lower in OPN-gKO or OPN-cKO mice. HBEs transferred with OPN enhanced the expression of phosphoinositide 3-kinase (PI3K)CA/p110α, PIK3CB/p110β, PIK3CD/p110δ, PIK3CG/p110γ, PIK3R1, PIK3R2 or PIK3R3. Spatial locations of OPN and OPN-positive epithelial subtypes showed the tight contact of airway epithelia and TCs. Epithelial OPN regulated the epithelial communication with TCs, and the down-regulation of OPN induced more alterations in transcriptomic profiles than the up-regulation. Our data evidenced that OPN regulated lung epithelial inflammation, injury, and cell communication between epithelium and TCs in acute and chronic lung injury. The conditional control of lung epithelial OPN may be an alternative for preventing and treating epithelial-origin lung inflammation and injury.

First report of primary testicular leiomyosarcoma in two dogs

BackgroundTesticular tumours are common in dogs and, among them, interstitial cell tumours, seminomas and sustentacular cell tumours are the most reported. Mesenchymal testicular tumours are rarely reported in humans as in veterinary medicine where only three cases of sarcomas (leiomyomas and leomyosarcomas) have been described in two stallions and in a ram.Case presentationThe present cases regarded a 12-year-old mixed-breed dog and a 10-year-old American Staffordshire Terrier that underwent bilateral orchiectomy. Formalin fixed testes were referred for histopathological diagnosis. At gross examination, in one of the testes of both dogs, a white, firm and variably cystic testicular mass, effacing and replacing the testicular parenchyma was detected. Samples were collected from both neoplastic and contralateral testes, routinely processed for histology and serial sections were also examined immunohistochemically with primary antibodies against cytokeratins, vimentin, Von Willebrand factor, inhibin-α, α-smooth muscle actin, smooth muscle myosin and desmin. Histopathological features as well as the immunohistochemical results, positive for vimentin, actin, myosin and desmin, confirmed the mesenchymal origin and the myoid phenotype of both testicular tumours supporting the diagnoses of leiomyosarcoma.ConclusionsTo the authors knowledge these are the first cases of primary testicular sarcoma reported in the canine species. However, even rare, these tumours deserve to be considered in routine diagnosis when a testicular spindle cell tumour is observed. The immunohistochemical panel applied was useful to distinguish the present tumours from undifferentiated Sertoli cell tumours confirming the diagnosis of leiomyosarcoma.

Urinary Modified C-Reactive Protein is Closely Associated with Tubulointerstitial Lesions in Lupus Nephritis.

Modified C-reactive protein (mCRP) is known to be involved in the upregulation and amplification of the local inflammatory response. This study investigated the circulating and local levels of mCRP and their relevance to clinicopathological features in patients with lupus nephritis. Ninety-five patients with renal biopsy-proven lupus nephritis and 30 normal controls were enrolled in this study. Plasma and urinary mCRP were screened by enzyme-linked immunosorbent assay (ELISA). The renal deposition of mCRP was detected by immunohistochemistry and immunofluorescence staining. A human proximal tubular epithelial cell line (HK2 cells) was incubated with purified IgG from lupus nephritis, and the production of CRP by HK2 cells was further evaluated. Plasma and urinary levels of mCRP increased significantly in patients with lupus nephritis compared with normal controls (P = 0.013, P < 0.001, respectively). The urinary mCRP levels were associated with interstitial inflammatory cell infiltration (r = 0.514, P < 0.001) and interstitial fibrosis (r = 0.270, P = 0.008). The ROC-AUC of the urinary mCRP levels for diagnosing tubulointerstitial lesions was 0.766. The urinary mCRP levels were closely associated with poor outcomes (HR: 1.204, 95% CI: 1.029-1.409, P = 0.020). However, no correlations were found of the plasma mCRP levels with clinicopathological data or the prognosis of lupus nephritis. CRP was mostly deposited in the renal tubules in patients with lupus nephritis, and the expression of CRP was significantly correlated with tubulointerstitial lesion indices. Immunofluorescence staining showed that mCRP could colocalize with IgG in tubules. Lupus nephritis-derived IgG could induce CRP production by HK2 cells. Urinary mCRP levels were significantly increased, and urinary mCRP might be a biomarker for tubulointerstitial lesions in patients with lupus nephritis. Renal CRP could be produced by tubular epithelial cells after stimulation by lupus nephritis-derived IgG, and the local presence of mCRP might play a critical role in the development of tubulointerstitial lesions.

Sensitivity analysis of a multi-physics model for the vascular microenvironment.

The vascular microenvironment is the scale at which microvascular transport, interstitial tissue propertiesand cell metabolism interact. The vascular microenvironment has been widely studied by means of quantitative approaches, including multi-physics mathematical models as it is a central system for the pathophysiology of many diseases, such as cancer. The microvascular architecture is a key factor for fluid balance and mass transfer in the vascular microenvironment, together with the physical parameters characterizing the vascular wall and the interstitial tissue. The scientific literature of this field has witnessed a long debate about which factor of this multifaceted system is the most relevant. The purpose of this work is to combine the interpretative power of an advanced multi-physics model of the vascular microenvironment with state of the art and robust sensitivity analysis methods, in order to determine the factors that most significantly impact quantities of interest, related in particular to cancer treatment. We are particularly interested in comparing the factors related to the microvascular architecture with the ones affecting the physics of microvascular transport. Ultimately, this work will provide further insight into how the vascular microenvironment affects cancer therapies, such as chemotherapy, radiotherapyor immunotherapy.

Adaptation strategies of juvenile grass carp (Ctenopharyngodon idella) facing different dissolved oxygen concentrations in a recirculating aquaculture system

Fishes exposed to different dissolved oxygen concentrations over prolonged time periods have different susceptibility and adaptation mechanisms. In this study, healthy grass carp with an average weight of 214.6 ​± ​18.7 ​g were selected for long-term culture in recirculating aquaculture systems at three different dissolved oxygen concentrations, i.e., 3.00 ​mg/L [(2.57 ​± ​0.33) mg/L, DO2.57), 5.00 ​mg/L [(4.61 ​± ​0.11) mg/L, DO4.61], which was the control group, and 7.00 ​mg/L [(6.50 ​± ​0.48) mg/L, DO6.50]. Blood, gill, muscle, and liver samples were collected after 60 ​days. Using hematoxylin-eosin (HE), alcian blue periodic acid Schiff (AB-PAS) staining, determination of respiratory metabolism enzyme activity, and fluorescent quantitative PCR, the adaptability of juvenile grass carp facing different dissolved oxygen levels were explored. The results showed that the respiration rate increased, and hemoconcentration and hemoglobin (Hb), electrolyte and pH changed at DO2.57. Furthermore, gill lamellae became thinner and longer, the distance between them increased, the mucus on them decreased and the interstitial cell mass decreased. In addition, the relative expression of apoptosis-related genes bcl-2 and bcl-xl in gill tissue decreased, while caspase-3 and bax increased. In muscles and liver tissues, lactate dehydrogenase (LDH) activity and lactic acid content (LA) increased, pyruvate dehydrogenase (PDH) activity decreased, and genes related to the respiratory metabolic enzymes PDH-A4 and PDH-E1α also changed accordingly. The oxygen consumption rate decreased significantly (P ​< ​0.05). At DO6.50, the distance between gill lamellae, numbers of red blood cells (RBC) and the content of hemoglobin decreased wheras the mucus on gill lamellae increased. The activity of PDH, the expression of PDH-E1a in liver and oxygen consumption rate increased. Different dissolved oxygen concentrations affected the respiratory behavior, gill tissue structure, blood transport capacity and tissue enzyme activity of juvenile grass carp. In summary, juvenile grass carp showed different adaptation strategies when facing different dissolved oxygen concentrations over prolonged periods in recirculating aquaculture systems.

Gypenosides suppress fibrosis of the renal NRK-49F cells by targeting miR-378a-5p through the PI3K/AKT signaling pathway.

The incidence of renal fibrosis caused by chronic kidney disease is increasing year by year. Preventing the activation and conversion of kidney-intrinsic fibroblasts to a myofibroblast phenotype is an important target for blocking the development of renal interstitial fibrosis. Our team established a stable renal interstitial fibrosis cell model in the early stage, and the screening results showed that GPs has good anti-fibrosis potential. At this stage, only a few literatures have reported its anti-fibrosis effect, and the mechanism of action is still unclear. The massive synthesis and secretion of extracellular-matrix (ECM) components by activated fibroblasts in the kidneys causes irreversible renal interstitial fibrosis. Gypenosides (GPs) have been shown to decelerate this process, in which micro RNAs (miRNAs) play an important regulatory role. This study aimed to evaluate the mechanism underlying the suppressive effect of GPs on renal fibrosis. This study used TGF-β1-stimulated NRK-49F renal cells as an in-vitro model of renal interstitial fibrosis. First, the concentration range of GPs that significantly affects the cytoactive was determined. Then, the anti-fibrotic effects of various concentrations of GPs in the in-vitro model were assessed via immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Non-coding-RNA sequencing combined with bioinformatics was used to predict the mechanistic basis of the anti-fibrotic effect of GPs, and qRT-PCR was used to verify the sequencing results and bioinformatic predictions. The identified relationships of the anti-fibrotic effect of GPs with miR-378a-5p and the PI3K/AKT signaling were evaluated using a miR-NC mimic and the PI3K inhibitor LY294002 as controls, respectively. TGF-β1 stimulation up-regulated α-SMA, COL1, and COL3 in NRK-49F cells, and this effect was suppressed by GPs. Additionally, TGF-β1 stimulation significantly changed the expression levels of 151 miRNAs, and GPs significantly suppressed the effect of TGF-β1 on the levels of 18 of these miRNAs. Among them, miR-3588 and miR-378a-5p were down-regulated, and miR-135b-5p and miR-3068-5p were up-regulated upon TGF-β1 induction. Of these miRNAs, miR-378a-5p was predicted to target the mRNAs of numerous proteins mainly enriched in the PI3K/AKT signaling pathway. The miRNA transfection experiments with the miR-NC mimic and PI3K inhibitor as controls showed that miR-378a-5p overexpression could suppress the TGF-β1-induced up-regulation of α-SMA, COL1, PI3K, and AKT, including the phosphorylated form (p-AKT). GPs inhibit the PI3K/AKT signaling by up-regulating miR-378a-5p in TGF-β1-stimulated NRK-49F cells and thereby reduce their massive secretion of ECM components. Given that this in-vitro model of renal interstitial fibrosis closely mimics the in-vivo pathogenesis, our results most likely apply to the in-vivo conditions.

Three-Dimensional Bioprinting of Ovine Aortic Valve Endothelial and Interstitial Cells for the Development of Multicellular Tissue Engineered Tissue Constructs.

To investigate the pathogenic mechanisms of calcified aortic valve disease (CAVD), it is necessary to develop a new three-dimensional model that contains valvular interstitial cells (VIC) and valvular endothelial cells (VEC). For this purpose, ovine aortic valves were processed to isolate VIC and VEC that were dissolved in an alginate/gelatin hydrogel. A 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany) was used to print cell-laden tissue constructs containing VIC and VEC which were cultured for up to 21 days. The 3D-architecture, the composition of the culture medium, and the hydrogels were modified, and cell viability was assessed. The composition of the culture medium directly affected the cell viability of the multicellular tissue constructs. Co-culture of VIC and VEC with a mixture of 70% valvular interstitial cell and 30% valvular endothelial cell medium components reached the cell viability best tested with about 60% more living cells compared to pure valvular interstitial cell medium (p = 0.02). The tissue constructs retained comparable cell viability after 21 days (p = 0.90) with different 3D-architectures, including a "sandwich" and a "tube" design. Good long-term cell viability was confirmed even for thick multilayer multicellular tissue constructs. The 3D-bioprinting of multicellular tissue constructs with VEC and VIC is a successful new technique to design tissue constructs that mimic the structure of the native aortic valve for research applications of aortic valve pathologies.

Niclosamide Inhibits Aortic Valve Interstitial Cell Calcification by Interfering with the GSK-3β/β-Catenin Signaling Pathway.

The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.

High resolution monitoring of valvular interstitial cell driven pathomechanisms in procalcific environment using label-free impedance spectroscopy.

Fibro-calcific aortic valve disease has high prevalence and is associated with significant mortality. Fibrotic extracellular matrix (ECM) remodeling and calcific mineral deposition change the valvular microarchitecture and deteriorate valvular function. Valvular interstitial cells (VICs) in profibrotic or procalcifying environment are frequently used in vitro models. However, remodeling processes take several days to weeks to develop, even in vitro. Continuous monitoring by real-time impedance spectroscopy (EIS) may reveal new insights into this process. VIC-driven ECM remodeling stimulated by procalcifying (PM) or profibrotic medium (FM) was monitored by label-free EIS. Collagen secretion, matrix mineralization, viability, mitochondrial damage, myofibroblastic gene expression and cytoskeletal alterations were analyzed. EIS profiles of VICs in control medium (CM) and FM were comparable. PM reproducibly induced a specific, biphasic EIS profile. Phase 1 showed an initial impedance drop, which moderately correlated with decreasing collagen secretion (r = 0.67, p = 0.22), accompanied by mitochondrial membrane hyperpolarization and cell death. Phase 2 EIS signal increase was positively correlated with augmented ECM mineralization (r = 0.97, p = 0.008). VICs in PM decreased myofibroblastic gene expression (p < 0.001) and stress fiber assembly compared to CM. EIS revealed sex-specific differences. Male VICs showed higher proliferation and in PM EIS decrease in phase 1 was significantly pronounced compared to female VICs (male minimum: 7.4 ± 4.2%, female minimum: 26.5 ± 4.4%, p < 0.01). VICs in PM reproduced disease characteristics in vitro remarkably fast with significant impact of donor sex. PM suppressed myofibroblastogenesis and favored ECM mineralization. In summary, EIS represents an efficient, easy-to-use, high-content screening tool enabling patient-specific, subgroup- and temporal resolution.