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Reconstituted 2D Cell and Tissue Models
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Nicole Schneider-Daum, Patrick Carius, Justus C. Horstmann, Claus-Michael Lehr
While AT II cells normally show a restricted proliferation in vitro, Bove et al. (2014) could retain AT II cell characteristics by co-culturing primary human AT II cells together with feeder cells from mouse and simultaneously treating the cells with a Rho kinase inhibitor “Y-27632.” By this method, some epithelial barrier function with a TEER of ~350–400 Ω*cm2 could be established (Bove et al. 2014). Methods to primary isolate AT I cells have so far only been described for rat tissue (Borok et al. 2002; Chen et al. 2004; Gonzalez and Dobbs 2013). None of these methods, however, was intended for the generation of electrically tight barriers after in vitro cultivation. Interestingly enough, AT II cells can be trans-differentiated into AT I-like cells, forming monolayers with significant barrier properties when cultivated in vitro which was reported first for rats (Danto et al. 1995) and later for humans (Elbert et al. 1999; Fuchs et al. 2003). By further improving the protocol, isolated human alveolar epithelial cells (hAEpC) seeded on permeable supports can be trans-differentiated into AT I-like cells, developing tight intercellular junctions and reaching TEER values of ~2000–2500 Ω*cm2 within 1 week (Daum et al. 2012). Even though such procedures allow for transport and uptake studies, the poor availability of human tissue still limits their wider application.
Physiological Properties of the Lower Urinary Tract
Published in Anthony R. Mundy, John M. Fitzpatrick, David E. Neal, Nicholas J. R. George, The Scientific Basis of Urology, 2010
MLCP activity can also be reduced by phosphorylation, which increases the Ca2+ sensitivity of the contractile system. Of significance is inhibition of MLCP activity by rho-associated kinase (ROK or ROCK) (74), which in turn is activated by small G proteins of the rho-family. In detrusor two isoforms of ROCK (I and II) have been identified (74). Inhibitors of ROCK activity, such as Y-27632 and HA-1077, attenuate agonist-induced contractions, but do not affect depolarization-mediated [with high (KCl)] contractures (75), which suggests that the rho-kinase pathway plays a role in the contractile state of the bladder.
Red Blood Cell and Platelet Mechanics
Published in Michel R. Labrosse, Cardiovascular Mechanics, 2018
Clot retraction (contraction, or shrinking of a blood clot) is necessary to promote wound closure, secure hemostasis, and prevent thrombotic occlusion of a vessel (Figure 8.12b). It was reported that clot retraction (duration in an in vitro assay: 20–120 min) is a multistep process that can be divided into three phases: initiation of contraction, linear contraction, and mechanical stabilization (Tutwiler et al. 2016). In Phase 1, the initiation of clot contraction is mediated by platelet activation (~2 min), fibrin network formation, platelet–fibrin binding, and start of platelet contraction. Phase 2 consists of the continuation of the latter, along with fibrin network remodeling. Finally, fibrin crosslinking via coagulation factor XIIIa causes mechanical stabilization (Phase 3). Taken together, thrombin, high platelet counts, platelet–fibrin binding, fibrin crosslinking, and platelet contraction support clot retraction, whereas high fibrinogen concentration, high hematocrit, and increased RBC mechanical interference limit clot retraction (Tutwiler et al. 2016). The importance of platelets in clot retraction is corroborated by a study in which the elasticities of platelet-rich clots and platelet-free clots were determined to be 600 Pa and 70 Pa, respectively (Jen and McIntire 1982, Carr 2003). Single platelets can generate high contraction forces, ranging from 1.5 to 79 nN, form adhesions stronger than 70 nN, have an elasticity of 10 kPa after contraction, and show an extensibility mean of about 1.57 before rupture from a fibrinogen-coated surface (Lam et al. 2011). However, it has to be noted that different adhesion force values can be obtained, depending on the coated matrix (Nguyen et al. 2016). Platelets can generate higher stall forces when exposed to a stiffer microenvironment. Thus, platelets can stiffen fibrin fibers, which contribute to the stiffening of the whole clot (Figure 8.13) (Lam et al. 2011). Moreover, considerable evidence has accumulated, suggesting that the actin–myosin complex is a crucial component for clot compaction. Platelets treated with the cell-permeable and selective drug Y-27632 to inhibit the Rho-associated, coiled-coil-containing protein kinase (ROCK), ML-7 to inhibit the myosin light-chain kinase (MLCK), and blebbistatin to inhibit the myosin ATPase activity generated strongly reduced platelet forces, as quantified by deflection of microposts (Feghhi et al. 2016). Similar observations were made using platelets from patients with the bleeding disorders Wiskott–Aldrich syndrome (WAS) and MYH9-related disease, in which the platelet cytoskeletal machinery is affected. Here, significantly lower contraction forces on soft and stiff environments were measured for platelets from patients than for platelets from healthy volunteers. Interestingly, a larger subpopulation of platelets from these patients showed almost no contractile force on the stiff environment (Myers et al. 2017). These data suggest that defects in mechanical properties of platelets can translate into increased bleeding risk.
Targeting the LPA1 signaling pathway for fibrosis therapy: a patent review (2010-present)
Published in Expert Opinion on Therapeutic Patents, 2022
Zhihao Gu, Yong Yan, Hequan Yao, Kejiang Lin, Xuanyi Li
Y-27632 treats liver fibrosis by inhibiting ROCK activity to prohibit the proliferation and migration of liver fibroblasts. However, Y-27632 causes vascular smooth muscle relaxation, which leads to the side effect of the sharp decrease in blood pressure. Moreover, Y-27632 easily decomposes in mammals, and thus larger doses are required to maintain an effective concentration, which further increases the potential toxicity of the drug [161,162]. Wu, H. et al. from Jiangsu Tasly Diyi Pharmaceutical Co., Ltd. modified Y-27632 into a prodrug to increase stability and reduce side effects. By connecting Y-27632 with a phosphate group, compound 1,076,762 had a phospholipid or phosphoramide scaffold. Compared with Y-27632, compound 1,076,762 had low polarity and increased membrane permeability, which improved the stability in other organs and tissues. When entering hepatic stellate cells, it is hydrolyzed by hydrolase in the liver to release the active molecule Y-27632, thereby playing a role in treating liver fibrosis. ELISAs showed reduced serum laminin (LN), type I collagen (CI), precollagen III (PC-III) and type IV collagen (C-IV) levels, indicating the suppression of liver fibrosis in SD rats [163].
RhoA/ROCK Signaling Regulates TGF-β1-Induced Fibrotic Effects in Human Pterygium Fibroblasts through MRTF-A
Published in Current Eye Research, 2022
Jiajun Xie, Qingyao Ning, Huina Zhang, Shuang Ni, Juan Ye
After TGF-β1 treatment, the RhoA/ROCK signaling activation in HPFs was increased as revealed by the expression of ROCK1 (2.34 ± 0.29 folds of mRNA, P < .0024, and 1.50 ± 0.04 folds of protein, P < .0001, respectively) and RhoA (3.18 ± 0.33 folds of mRNA, P = .0004, and 2.40 ± 0.04 folds of protein, P < .0001, respectively), comparing with the control group (Figure 3a–e). In contrast, the mRNA and protein expression of ROCK1 and RhoA induced by TGF-β1 were reduced in the presence of Y-27632. Particularly, TGF-β1-induced expression ROCK1 and RhoA mRNA was effectively inhibited by Y-27632 at the selected concentration of 5 μM (~0.6 fold compared to TGF-β1 group) and 10 μM Y-27632 (~0.5 fold compared to TGF-β1 group) as shown in Figure 3a,b.
Regenerative responses of rabbit corneal endothelial cells to stimulation by fibroblast growth factor 1 (FGF1) derivatives, TTHX1001 and TTHX1114
Published in Growth Factors, 2021
Jessica Weant, David D. Eveleth, Amuthakannan Subramaniam, Jennifer Jenkins-Eveleth, Michael Blaber, Ling Li, David M. Ornitz, Asaf Alimardanov, Trevor Broadt, Hui Dong, Vinay Vyas, Xiaoyi Yang, Ralph A. Bradshaw
FGF1 and its analogs are not the only agents that may impact the proliferation and migration of CECs. Other growth factors (FGF2, EGF, etc) (Thalmann-Goetsch, Engelmann, and Bednarz 1997) as well as small molecules including p38 MAP kinase inhibitors (Nakahara et al. 2018) and Rho kinase inhibitors (Schlötzer-Schrehardt et al. 2021; Okumura et al. 2016) have been reported to stimulate proliferation and or migration. The Rho kinase inhibitor netarsudil has been clinically tested in humans and does not appear to impact CEC density after 6 months of treatment (Price, Feng, and Price 2021) although it has some effect on corneal thickness in FECD patients (Price and Price 2021). The reported proliferative effects of Rho kinase inhibitors on CECs in vitro are not large and the effect of Y-27632 is not reproducible in all laboratories (Meekins et al. 2016), while the proliferative effects of FGF1 in vitro are substantial and have been reproduced in several laboratories. In a direct comparison of stimulation of migration in vitro, FGF2 stimulated migration more strongly than Y-27632 (Lee and Kay 2006). Currently, a number of investigators are examining the impact of ripasudil on CEC migration in the context of Descemet’s stripping only surgery (DSO) as an alternative to transplantation (Macsai and Shiloach 2019; Moloney et al. 2021). While there is not a direct comparison of FGF1 to Rho kinase inhibitors in vitro or in vivo, comparison of the magnitude of the effects in separate experiments suggests that FGF1 is at least as effective as Rho kinase inhibitors and may potentially be more so, particularly in stimulating proliferation.