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Von Willebrand Factor (VWF) can promote platelet adhesion to the post-atherosclerotic regions to initiate thrombosis. The synthesis and secretion of VWF are important functions of endothelial cells (ECs). However, the mechanism through which blood flow regulates endothelial secretion of VWF remains unclear. We utilized a parallel-plate flow apparatus to apply fluid shear stress to human umbilical vein endothelial cells (HUVECs). Compared with pulsatile shear stress that mimics laminar flow in the straight parts of arteries or upstream of atherosclerotic stenosis sites, short-term exposure to oscillatory shear stress (OS) that mimics disturbed flow increased VWF secretion independent of affecting synaptosomal-associated protein 23 (SNAP23) expression and promoted the translocation of SNAP23 to the cell membrane. Vimentin associated with SNAP23, and this association was enhanced by OS or histamine. Acrylamide, a reagent that disrupts vimentin intermediate filaments, prevented histamine/OS-induced SNAP23 translocation, as well as VWF secretion. Immunofluorescence analysis revealed that the polarity of the vimentin intermediate filament network decreased after stimulation with histamine or OS. In addition, inhibition of protein kinase A (PKA) or G protein coupled receptor 68 (GPR68) eliminated the histamine/OS-induced phosphorylation of vimentin at Ser38 and secretion of VWF. Furthermore, syntaxin 7 might assist with the translocation of SNAP23 to the cell membrane, thus playing a role in promoting VWF secretion. The GPR68/PKA/vimentin signaling pathway may represent a novel mechanism for the regulation of SNAP23-mediated VWF secretion by ECs under OS and provide strategies for the prevention of atherosclerosis-related thrombosis.

Introduction: As a comprehensive sport, Marathon presents high demands concerning the athletes’ comprehensive capacity. Objective: This paper explores to what extent balance skill training can effectively contribute to improving physical fitness for marathon runners. Methods: 120 marathon runners were selected for the pilot experiment. The experimental group received balance training, while the control group underwent no intervention. Results: In the experimental group, the time with eyes closed and feet apart increased from 33.559 ± 15.8570 to 37.203 ± 15.5865s, the time spent in the T-shaped run from 10.144 ± 0.5063s to 9.908 ± 0.5225s, the time spent in the standing long jump from 2.831 ± 0.2648m to 3.058 ± 0.3183m, and the time spent in the T-shaped run from 55.544 ± 2.2581 to 60.845 ± 2.4367 times/min. The total FMS score changed from 14.618 ± 0.9392 to 18.481 ± 1.3909. Shoulder flexibility increased from 2.777 ± 0.4756 to 2.917 ± 0.2994, and active straight knee raise changed from 2.306 ± 0.4692 to 2.803 ± 0.403. Conclusion: Daily balance training can be added to usual marathon training to improve the fitness of its athletes. Level of evidence II; Therapeutic studies - investigation of treatment outcomes. RESUMO Introdução: A maratona, enquanto esporte integral, apresenta altas exigências relativas à capacidade plena dos atletas. Objetivo: Este artigo tenta explorar em que medida o treinamento da habilidade de equilíbrio pode efetivamente contribuir para o aprimoramento da aptidão física aos praticantes do esporte de corrida de maratona. Métodos: 120 corredores de maratona foram selecionados para o experimento piloto. O grupo experimental recebeu treinamento de equilíbrio, enquanto o grupo de controle não sofreu qualquer intervenção. Resultados: No grupo experimental, o tempo com olhos fechados e pés separados aumentou de 33,559 ± 15,8570 para 37,203 ± 15,5865s, o tempo gasto na corrida em forma de T de 10,144 ± 0,5063s para 9,908 ± 0,5225s, o tempo gasto no salto em pé em distância de 2,831 ± 0,2648m para 3,058 ± 0,3183m, e o tempo gasto na corrida em forma de T de 55,544 ± 2,2581 para 60,845 ± 2,4367 vezes/min. A pontuação total de FMS mudou de 14,618 ± 0,9392 para 18,481 ± 1,3909. A flexibilidade do ombro aumentou de 2,777 ± 0,4756 para 2,917 ± 0,2994, e o levantamento reto do joelho ativo mudou de 2,306 ± 0,4692 para 2,803 ± 0,403. Conclusão: O treinamento de equilíbrio diário pode ser adicionado ao treino de maratona habitual para melhorar a aptidão física de seus atletas. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento. RESUMEN Introducción: El maratón, como deporte integral, presenta grandes exigencias en cuanto a la capacidad integral de los atletas. Objetivo: Este trabajo pretende explorar hasta qué punto el entrenamiento de las habilidades de equilibrio puede contribuir eficazmente a la mejora de la condición física de los corredores de maratón. Métodos: Se seleccionaron 120 corredores de maratón para el experimento piloto. El grupo experimental recibió entrenamiento de equilibrio, mientras que el grupo de control no se sometió a ninguna intervención. Resultados: En el grupo experimental, el tiempo con los ojos cerrados y los pies separados aumentó de 33,559 ± 15,8570 a 37,203 ± 15,5865s, el tiempo empleado en la carrera en forma de T de 10,144 ± 0,5063s a 9,908 ± 0,5225s, el tiempo empleado en el salto de longitud de pie de 2,831 ± 0,2648m a 3,058 ± 0,3183m, y el tiempo empleado en la carrera en forma de T de 55,544 ± 2,2581 a 60,845 ± 2,4367 veces/min. La puntuación total de FMS pasó de 14,618 ± 0,9392 a 18,481 ± 1,3909. La flexibilidad de los hombros aumentó de 2,777 ± 0,4756 a 2,917 ± 0,2994, y la elevación activa de la rodilla pasó de 2,306 ± 0,4692 a 2,803 ± 0,403. Conclusión: El entrenamiento diario del equilibrio puede añadirse al entrenamiento habitual de maratón para mejorar la forma física de sus atletas. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Disturbed blood flow induces endothelial pro-inflammatory responses that promote atherogenesis. Nanoparticle-based therapeutics aimed at treating endothelial inflammation in vasculature where disturbed flow occurs may provide a promising avenue to prevent atherosclerosis. By using a vertical-step flow apparatus and a microfluidic chip of vascular stenosis, herein, it is found that the disk-shaped versus the spherical nanoparticles exhibit preferential margination (localization and adhesion) to the regions with the pro-atherogenic disturbed flow. By employing a mouse model of carotid partial ligation, superior targeting and higher accumulation of the disk-shaped particles are also demonstrated within disturbed flow areas than that of the spherical particles. In hyperlipidemia mice, administration of disk-shaped particles loaded with hypomethylating agent decitabine (DAC) displays greater anti-inflammatory and anti-atherosclerotic effects compared with that of the spherical counterparts and exhibits reduced toxicity than "naked" DAC. The findings suggest that shaping nanoparticles to disk is an effective strategy for promoting their delivery to atheroprone endothelia.

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

Vascular smooth muscle cell (vSMC) is highly plastic as its phenotype can change in response to mechanical cues inherent to the extracellular matrix (ECM). VSMC may be activated from its quiescent contractile phenotype to a proinflammatory phenotype, whereby the cell secretes chemotactic and inflammatory cytokines, e.g. MCP1 and IL6, to functionally regulate monocyte and macrophage infiltration during the development of various vascular diseases including arteriosclerosis. Here, by culturing vSMCs on polyacrylamide (PA) substrates with variable elastic moduli, we discovered a role of discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds collagens, in mediating the mechanical regulation of vSMC gene expression, phenotype, and proinflammatory responses. We found that ECM stiffness induced DDR1 phosphorylation, oligomerization, and endocytosis to repress the expression of DNA methyltransferase 1 (DNMT1), very likely in a collagen-independent manner. The DDR1-to-DNMT1 signaling was sequentially mediated by the extracellular signal-regulated kinases (ERKs) and p53 pathways. ECM stiffness primed vSMC to a proinflammatory phenotype and this regulation was diminished by DDR1 inhibition. In agreement with the in vitro findings, increased DDR1 phosphorylation was observed in human arterial stiffening. DDR1 inhibition in mouse attenuated the acute injury or adenine diet-induced vascular stiffening and inflammation. Furthermore, mouse vasculature with SMC-specific deletion of Dnmt1 exhibited proinflammatory and stiffening phenotypes. Our study demonstrates a role of SMC DDR1 in perceiving the mechanical microenvironments and down-regulating expression of DNMT1 to result in vascular pathologies and has potential implications for optimization of engineering artificial vascular grafts and vascular networks.

Vascular smooth muscle cells (SMCs) can adapt to changes in cellular geometric cues; however, the underlying mechanisms remain elusive. Using 2D micropatterned substrates to engineer cell geometry, it is found that in comparison with an elongated geometry, a square-shaped geometry causes the nuclear-to-cytoplasmic redistribution of DNA methyltransferase 1 (DNMT1), hypermethylation of mitochondrial DNA (mtDNA), repression of mtDNA gene transcription, and impairment of mitochondrial function. Using irregularly arranged versus circumferentially aligned vascular grafts to control cell geometry in 3D growth, it is demonstrated that cell geometry, mtDNA methylation, and vessel contractility are closely related. DNMT1 redistribution is found to be dependent on the phosphoinositide 3-kinase and protein kinase B (AKT) signaling pathways. Cell elongation activates cytosolic phospholipase A2, a nuclear mechanosensor that, when inhibited, hinders AKT phosphorylation, DNMT1 nuclear accumulation, and energy production. The findings of this study provide insights into the effects of cell geometry on SMC function and its potential implications in the optimization of vascular grafts.

Macrophage-mediated inflammatory responses occur throughout all stages of atherosclerosis. DNA methylation is one of the critical epigenetic mechanisms and is associated with the development of atherosclerosis. The underlying mechanism of epigenetic regulation of macrophage inflammation (M1 activation) remains unclear. Here we aim to study the role of DNA methyltransferase 1 (DNMT1) in modulating macrophage inflammation and atherosclerosis. DNMT1 expression is up-regulated in THP-1-derived macrophages upon treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overexpression of DNMT1 promotes the LPS- and IFN-γ-induced M1 activation whereas inhibition of DNMT1 attenuates it. Consistently, DNMT1 expression is elevated in macrophages in atherosclerotic plaques from human and mouse specimens; compared with the Dnmt1wild-type, myeloid Dnmt1 deficiency in mice in an Apolipoprotein E (ApoE) knockout background or receiving AAV-PSCK9 injection and carotid partial ligation results in ameliorated atheroma formation and suppressed plaque inflammation. The promoter regions of atheroprotective Krüppel-like factor 4 (KLF4) are hypermethylated in M1- activated macrophages. DNMT1 down-regulates the expression of KLF4, probably through catalyzing DNA methylation of the promoter regions of KLF4. Gain- and loss-of function study of KLF4 indicates that the DNMT1-mediated macrophage M1 activation is dependent on KLF4. Our data demonstrate a proatherogenic role for DNMT1 as a defining factor in macrophage inflammation both in vitro and in vivo. DNMT1 promotes macrophage M1 activation by suppressing KLF4 expression. Thus macrophage-specific DNMT1 inhibition may provide an attractive therapeutic potential to prevent or reduce atherosclerosis.

Background: Hyperuricemia causes gout syndrome, which can manifest as hemorheological damage. However, hemorheological properties have not been well characterized in asymptomatic conditions. Aims: The present study was aimed to investigate the hemorheological changes in rats with asymptomatic hyperuricemia. Methods and Materials: The rat model was established by intraperitoneal injection of 250 mg/(kg·d) oxonate acid for 8 weeks. In one group of rats, 70 mg/(kg·d) allopurinol was also administrated for the latter 4 weeks. Control rats were administered CMCNa solution and water. Results: Hyperuricemic rats had higher blood viscosity than control rats did. Moreover, their erythrocytes had reduced deformation index, orientation index, surface charges, and osmotic fragility. Treatment with allopurinol decreased the blood uric acid amounts and improved the hemorheological parameters. Conclusions: Our study suggests that there is deterioration in the hemorheology of hyperuricemic rats and that allopurinol can play a protective role on the deterioration. Therefore, hemorheology can be used as a diagnostic tool in asymptomatic hyperuricemia.

Chinese dragon’s blood, the red resin ofDracaena cochinchinensis, one of the renowned traditional medicines, has been used to facilitate blood circulation and disperse blood stasis for thousands of years. Phenolic compounds are considered to be responsible for its main biological activities. In this study, total phenolic compounds of Chinese dragon’s blood were made into capsule (Longxuetongluo Capsule, LTC) and their effects on the abnormal hemorheological properties were examined by high fat diet (HFD) induced ApoE−/− mice. Compared to the model group, LTC recovered the abnormal hemorheological parameters in HFD-induced ApoE−/− mice by reducing whole blood viscosity (WBV) at high rate and improving erythrocyte function. In conclusion, LTC could ameliorate erythrocyte deformability and osmotic fragility through the reduction of lipid peroxidation on plasma and erythrocyte membranes in HFD-induced ApoE−/− mice, which supported the traditional uses of Chinese dragon’s blood as an effective agent for improving blood microcirculation in hypercholesterolemia.

(1) Background: There are no successive treatments for heart failure with preserved ejection fraction (HFpEF) because of complex interactions between environmental, histological, and genetic risk factors. The objective of the study is to investigate changes in cardiomyocytes and molecular networks associated with HFpEF. (2) Methods: Dahl salt-sensitive (DSS) rats developed HFpEF when fed with a high-salt (HS) diet for 7 weeks, which was confirmed by in vivo and ex vivo measurements. Shotgun proteomics, microarray, Western blot, and quantitative RT-PCR analyses were further carried out to investigate cellular and molecular mechanisms. (3) Results: Rats with HFpEF showed diastolic dysfunction, impaired systolic function, and prolonged repolarization of myocytes, owing to an increase in cell size and apoptosis of myocytes. Heatmap of multi-omics further showed significant differences between rats with HFpEF and controls. Gene Set Enrichment Analysis (GSEA) of multi-omics revealed genetic risk factors involved in cardiac muscle contraction, proteasome, B cell receptor signaling, and p53 signaling pathway. Gene Ontology (GO) analysis of multi-omics showed the inflammatory response and mitochondrial fission as top biological processes that may deteriorate myocyte stiffening. GO analysis of protein-to-protein network indicated cytoskeleton protein, cell fraction, enzyme binding, and ATP binding as the top enriched molecular functions. Western blot validated upregulated Mff and Itga9 and downregulated Map1lc3a in the HS group, which likely contributed to accumulation of aberrant mitochondria to increase ROS and elevation of myocyte stiffness, and subsequent contractile dysfunction and myocardial apoptosis. (4) Conclusions: Multi-omics analysis revealed multiple pathways associated with HFpEF. This study shows insight into molecular mechanisms for the development of HFpEF and may provide potential targets for the treatment of HFpEF.

The earliest atherosclerotic lesions preferentially develop in arterial regions experienced disturbed blood flow, which induces endothelial expression of pro-atherogenic genes and the subsequent endothelial dysfunction. Our previous study has demonstrated an up-regulation of DNA methyltransferase 1 (DNMT1) and a global hypermethylation in vascular endothelium subjected to disturbed flow. Here, we determined that DNMT1-specific inhibition in arterial wall ameliorates the disturbed flow-induced atherosclerosis through, at least in part, targeting cell cycle regulator cyclin A and connective tissue growth factor (CTGF). We identified the signaling pathways mediating the flow-induction of DNMT1. Inhibition of the mammalian target of rapamycin (mTOR) suppressed the DNMT1 up-regulation both in vitro and in vivo. Together, our results demonstrate that disturbed flow influences endothelial function and induces atherosclerosis in an mTOR/DNMT1-dependent manner. The conclusions obtained from this study might facilitate further evaluation of the epigenetic regulation of endothelial function during the pathological development of atherosclerosis and offer novel prevention and therapeutic targets of this disease.

Dendritic cells (DCs) are potent and specialized antigen-presenting cells that play a crucial role in initiating and amplifying both the innate and adaptive immune responses. Tumor cells can escape from immune attack by secreting suppressive cytokines which solely or cooperatively impair the immune function and microrheological properties of DCs. However, the underlying mechanisms are not fully defined. Transforming growth factor-β1 (TGF-β1) has been identified as a major cytokine in the tumor microenvironment. To determine the effects of TGF-β1 on mature DCs (mDCs) from microrheological viewpoint, cells were treated with different concentrations of TGF-β1. The results showed that the impaired microrheological parameters, including osmotic fragility, electrophoretic mobility, deformability, membrane fluidity, F-actin organization and so on, as well as motilities of mDCs relied heavily on TGF-β1 concentration. Moreover, these changes were correlated with the expression levels of fascin1, cofilin1, phosphorylated cofilin1 and profilin, this could be one of the crucial aspects of immune escape mechanisms of tumors, hinting that the signal pathway of TGF-β1 should be blocked in appropriate way before performing DCs-based immunotherapy against cancer. It is clinically important to understand the biological behavior of DCs and immune escape mechanism of tumor as well as how to improve efficiency of the anti-tumor therapy based on DCs.

The distal tubule and collecting duct in kidney regulate water homeostasis. TMOD1 is an actin capping protein that plays an important role in controlling the organization of actin filaments. In this study, we found TMOD1 was specifically expressed in distal tubules and collecting ducts. To investigate the role of TMOD1, we created Tmod1flox/flox mice and bred them with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice, Tmod1flox/flox/Ksp-Cre+ (designated as TFK). As compared with control mice, TFK mice showed oliguria, hyperosmolality urine, and high blood pressure. To determine the mechanisms underlying this phenotype, we performed label-free quantitative proteomics on kidneys of TFK and control mice. Total of 83 proteins were found differentially expressed. Bioinformatic analysis indicated that biological processes, including protein phosphorylation and metabolic process, were involved in TMOD1 regulatory network. Gene set enrichment analysis showed that multiple pathways, such as phosphatidylinositol signaling system and GnRH signaling pathway, were strongly associated with Tmod1 knockout. Western blot validated the down-regulation of three proteins, TGFBR2, SLC25A11, and MTFP1, in kidneys of TFK mice. Our study provides valuable information on the molecular functions and the regulatory network of Tmod1 gene in kidney, as well as the new mechanisms for the regulation of water balance.

Objective Urea transporter-B (UT-B), highly expressed in erythrocyte, confers specific permeability to urea, urea analogues and water. The purpose of this study was to determine the hemorheological properties of UT-B null erythrocyte using a series of biophysical techniques. Methods and results The blood was taken from UT-B knockout and wild-type mice and the hemorheological parameters were measured. The UT-B inhibitor, PU-14, was used to treat the erythrocyte of wild-type mice in vitro and the deformability of the treated erythrocyte was analyzed. The results showed that UT-B knockout improves the hemorheological properties of erythrocyte, including increased erythrocyte deformation index, small deformation index, orientation index, low osmotic fragility and high electrophoretic rate. The UT-B inhibitor PU-14 had the similar effect as UT-B knockout on the deformation indices. The whole blood viscosity in UT-B knockout mice showed reduction trend as compared to wild-type mice. Conclusions The data indicate that UT-B is involved in the regulation of hemorheology, which suggests that UT-B may be a potential therapeutic target for improving the hemorheology in some metabolic and hereditary diseases.

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors.

Hematopoietic stem cells (HSCs) are tissue specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Recently, people have learned a lot about the embryonic HSCs on their development and homing. During their differentiation, HSCs are regulated by the transcription factors, such as Runx1 and Notch signaling pathway, etc. MicroRNAs also regulate the self-renewal and differentiation of hematopoietic stem/progenitor cells on the post-transcriptional levels. Since the onset of circulation, the formation of HSCs and their differentiation into blood cells, especially red blood cells, are regulated by the hemodynamic forces. It would be of great significance if we could treat hematologic diseases with induced HSCs in vitro on the basis of fully understanding of hemotopoietic stem cell development. This review is focused on the advances in the research of HSCs' development and regulation.

We previously isolated a sulfated polysaccharide-protein complex from Gekko swinhonis Guenther, a traditional Chinese medicine, and have demonstrated its direct anti-cancer effect on human hepatocellular carcinoma cell line SMMC-7721. Here we investigated the effects of Gekko sulfated polysaccharide-protein complex (GSPP) on the defective biorheological characters of dendritic cells (DCs) under SMMC-7721 microenvironment. Our findings have shown that the biorheological properties of DCs were severely impaired by SMMC-7721 microenvironment, including decreased cell deformability, migration, and electrophoresis mobility, increased osmotic fragilities, and changed organizations of cytoskeletal proteins. We also found decreased secretion of interleukin (IL)-12 and increased secretion of IL-10 in DCs. However, supernatant collected from nonmalignant liver cells had no effect on these parameters. SMMC-7721 cells were treated with GSPP and the supernatant was used to culture DCs. We found that the defective biorheological parameters of DCs, except for osmotic fragility, were partially or completely improved. The secretion of IL-12 did not change as compared with that of DCs in SMMC-7721 microenvironment, but the secretion of IL-10 was resumed to the control level. Our results indicate that GSPP could partially restore the defective biorheological characteristics of DCs via modifying the tumor microenvironment and decreasing the secretion of IL-10 of DCs.

It has been shown that 90% of tumors, including hematological malignant tumors and leukemia, have much higher levels of telomerase expression than normal cells. To investigate the effect of telomerase on leukemia cells, we transfected K562, a human erythroleukemia cell line with an antisense-hTERT (human telomerase reverse transcriptase) cDNA vector, and examined the biological and biophysical properties of the stably transfected cells (referred to as KAT). Un-transfected cells (K562) and cells transfected with the empty vector (referred to as KC) were used as controls. Cell growth curve and (3)H-TdR test showed that the growth rate and DNA synthesis of KAT decreased compared with those of K562 and KC cells. Apoptosis and cell cycle distribution in KAT cells under normal culture condition were similar to those of K562 and KC cells, but changed after serum deprivation. KAT cells had significantly different biophysical characteristics from K562 and KC in terms of cell electrophoresis, membrane fluidity, membrane fluidity, and viscoelasticity. Furthermore, the transendothelial migration rate of KAT was much lower than those of K562 and KC cells. Confocal microscopy showed that KAT cells had higher F-actin content, suggesting the reorganization of cytoskeleton. Flow cytometry analysis revealed a lowered intracellular calcium concentration and CD71 expression, explaining the high F-actin content in KAT cells. In conclusion, we found that the knockdown of hTERT in K562 cells changed their cytoskeleton and biophysical features, and reduced the cell migration.

Fenfluramine is prescribed either alone or in combination with phentermine as part of Fen-Phen, an anti-obesity medication. Fenfluramine was withdrawn from the US market in 1997 due to reports of heart valvular disease, pulmonary arterial hypertension, and cardiac fibrosis. Particularly, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives. The underlying mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. We reasoned that investigating drug-induced gene dysregulation would enhance our understanding of the fenfluramine-associated pathogenic mechanism of IPAH. Whole-genome gene expression profiles in fenfluramine-treated human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells (isolated from normal subjects) were compared with baseline expression in untreated cells. Fenfluramine treatment caused dysregulation in a substantial number of genes involved in a variety of pathways and biological processes. In addition to several common pathways and biological processes such as “MAPK signaling pathway,” “inflammation response,” and “calcium signaling pathway” shared between both cell types, pathways and biological processes such as “blood circulation,” “muscle system process,” and “immune response” were enriched among the dysregulated genes in PASMC. Pathways and biological processes such as those related to cell cycle, however, were enriched among the dysregulated genes in PAEC, indicating that fenfluramine could affect unique pathways (or differentially) in different types of pulmonary artery cells. While awaiting validation in a larger cohort, these results strongly suggested that fenfluramine could induce significant dysregulation of genes in multiple biological processes and pathways critical for normal pulmonary vascular functions and structure. The transcriptional and posttranscriptional changes in these genes may, therefore, contribute to the pathogenesis of fenfluramine-associated IPAH.

Dendritic cells (DCs), which are potent antigen presenting cells (APCs), are utilized to deliver the signals essential for the initiation of immune responses. In this study, we used an interdisciplinary approach to characterize the effect of K562 cells, a human chronic myeloid leukemia (CML) cell line, on the biomechanical characteristics and immune functions of DCs. When co-cultured with K562 cells, the biomechanical and immunological characteristics of immature DCs (imDCs) and mature DCs (mDCs) were severely impaired compared with controls. The changes include increased membrane viscoelasticity, reorganized cytoskeleton (F-actin), suppressed capability of antigen uptake, transendothelium migration, and activation of naïve T cells. In exploring the mechanisms of these changes, we identified several genes and proteins by microarray analysis and 2D gel electrophoresis. Changes were found in the cytoskeleton-related genes and proteins (such as cofilin1 and profilin1) and matrix-related genes and proteins (such as TIMP1 and MMP9). These findings provide a molecular basis for the biomechanical and immunological changes of DCs in response to K562 and may help to elucidate the mechanism for tumor immune escape.

Aim of the study Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Here we investigated the structural characterization and anti-cancer effects of sulfated polysaccharide–protein complex (GSPP) isolated from Gekko swinhonis Guenther. Materials and methods The structure of GSPP was characterized by high performance liquid chromatography, gas chromatography, gas chromatography–mass spectrometry, β-elimination reaction, and NMR spectroscopy. SMMC-7721 cells were used to assess the influence of GSPP on hepatocellular carcinoma. Cell proliferation and survival was determined by trypan blue exclusion assay. Cell migration was performed by wound-healing and transwell assay. The secretion of IL-8 was detected by an enzyme-linked immunosorbent assay kit. Flow cytometry was used to analyze intracellular calcium concentration, as well as cell cycle distribution and apoptosis. Confocal microscopy was used to assess the localization and configuration of actin filaments. Results GSPP was chemically characterized as a sulfated polysaccharide–protein complex with O-glycopeptide linkages. Our results showed that GSPP inhibited the proliferation of SMMC-7721 cells and blocked cells in the S phase. No direct toxicity against cells was observed. Furthermore, GSPP inhibited the migration of SMMC-7721 cells with the reduction of intracellular calcium. Actin filaments were polymerized and accumulated in the cytoplasm of the treated cells, whereas the secretion of IL-8 was not significantly changed after GSPP exposure. Conclusion We describe an identified sulfated polysaccharide–protein complex, and demonstrate its direct effect on hepatocellular carcinoma cell migration via calcium-mediated regulation of the actin cytoskeleton reorganization.

Dichlorvos is an important insecticide used largely. Some studies have demonstrated that organophosphate pesticide has effects on erythrocyte membrane structures, which is critical to erythrocyte function and hemorheology. The aim of the present study was to explore the effect of oxidative stress on hemorheological changes during dichlorvos poisoning in rabbits. Data indicated that after dichlorvos exposure the hematocrit adjusted viscosity at high shear rate increased and erythrocyte membrane fluidity decreased. Data obtained from plasma showed that lipid peroxidative substance-malonaldehyde was elevated and superoxide dismutase was reduced. In summary, oxidative stress does occur in dichlorvos poisoning and may lead to hemorheological alterations. The changes of hemorheology may be responsible for the pathophysiology of the dichlorvos poisoning.

The differentiation, maturation and functioning of Dendritic cells (DCs) are dynamic processes. This study investigated the changes of DCs' migration ability and biorheological properties during their differentiation. Transmigration assay showed that, DCs' migration rate was improved significantly as they differentiate (p < 0.05); NSC (Rac1 blocker) treatment could significantly decrease their migration rates (p < 0.05). Confocal images showed that, F-actin uniformly distributed in monocytes; with DC's differentiation, F-actin began to remodel and gather at the site of dendrites; the images presented surface ruffles and uneven sawtooth-like cytoskeletal structures. Fluorescence polarization analysis showed that, membrane fluidity was increased significantly with DC's differentiation (p < 0.05). CD62L was upregulated significantly (p < 0.05) on the third and ninth days. CD2 was upregulated significantly (p < 0.05) until the seventh day. DC's electrophoretic mobility was increased continuously, especially increased significantly from the third day to the fifth day and the final stage (p < 0.05). These results indicate that there are significant changes in the biorheological properties of DCs during their differentiation.

Ryanodine receptors (RyRs) are a family of Ca2+ channel proteins that mediate the massive release of Ca2+ from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca2+ ([Ca2+](in)) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca2+](in) of RBCs. In the presence of RyR1, [Ca2+](in) showed biphasic changes, i.e., it increased with the extracellular Ca2+ ([Ca2+](ex)) up to 5muM and then decreased with the further increase of [Ca2+](ex). However, [Ca2+](in) remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca2+](in) mediated by RyR1 with the increases of [Ca2+](ex). Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca2+ influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca2+ release channel.

Pulmonary vascular remodeling occurs in patients with chronic thromboembolic pulmonary hypertension (CTEPH). One factor contributing to this vascular wall thickening is the proliferation of pulmonary artery smooth muscle cells (PASMC). Store-operated Ca2+ entry (SOCE) and cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC are known to be important in cell proliferation and vascular remodeling in pulmonary hypertension. Rapamycin is widely known for its antiproliferative effects in injured coronary arteries. Although several reports have suggested favorable effects of rapamycin in animal models of pulmonary hypertension, no reports have been published to date in human tissues. Here we report that rapamycin has an inhibitory effect on SOCE and an antiproliferative effect on PASMC derived from endarterectomized tissues of CTEPH patients. Cells were isolated from endarterectomized tissues obtained from patients undergoing pulmonary thromboendarterectomy (PTE). Immunohistochemical analysis indicated high deposition of platelet-derived growth factor (PDGF) in tissue sections from PTE tissues and increased PDGF receptor expression. PDGF transiently phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6 kinase in CTEPH cells from CTEPH patients. Acute treatment (30 min) with rapamycin (10 nM) slightly increased cyclopiazonic acid (10 μM)-induced Ca2+ mobilization and significantly reduced SOCE. Chronic treatment (24 h) with rapamycin reduced Ca2+ mobilization and markedly inhibited SOCE. The inhibitory effects of rapamycin on SOCE were less prominent in control cells. Rapamycin also significantly reduced PDGF-stimulated cell proliferation. In conclusion, the data from this study indicate the importance of the mTOR pathway in the development of pulmonary vascular remodeling in CTEPH and suggest a potential therapeutic benefit of rapamycin (or inhibition of mTOR) in these patients.

Pulmonary vascular remodeling, a major cause for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH), is partially due to increased proliferation of pulmonary arterial smooth muscle cells (PASMC) in the media, resulting in vascular wall thickening. Platelet-derived growth factor (PDGF) is a potent mitogen that may be involved in the progression of PAH. Blockade of PDGF receptors has been demonstrated to have therapeutic potential for patients with severe pulmonary hypertension. Prednisolone is an immunosuppressant shown to have anti-inflammatory and antiproliferative effects on PASMC. This study was designed to investigate whether PDGF and prednisolone affect human PASMC proliferation by regulating the nuclear translocation of NF-κB (a transcription factor composed of 2 subunits, p50 and p65). Treatment of human PASMC with PDGF (10 ng/ml) significantly increased nuclear translocation of p50 and p65 subunits. Inhibition of NF-κB activation or nuclear translocation of p50/p65 significantly attenuated PDGF-induced PASMC proliferation (determined by [3H]thymidine incorporation). In the presence of prednisolone (200 μM), the PDGF-induced nuclear translocation of p50 and p65 subunits was markedly inhibited ( P < 0.05 vs. the cells treated with PDGF alone). These results indicate that PDGF-induced nuclear translocation of NF-κB may play an important role in stimulating PASMC proliferation (and/or enhancing PASMC survival), whereas prednisolone may exert anti-inflammatory and antiproliferative effects on PASMC by inhibiting NF-κB nuclear translocation.

There is evidence that hyperlipidemia can induce hemorheological and microcirculatory disturbances. Myakuryu, a Chinese traditional medicine is efficacious in promoting lipid metabolism and protecting oxidative stress, but whether this drug can ameliorate rheologic disturbances caused by hyperlipidemia is still unknown. The present study was conducted to investigate the effects of myakuryu on hemorheological and microcirculatory disturbances induced by hyperlipidemia. Wistar rats were divided into a group on control diet (n=8) and a group on high-fat diet (HFD, n=44). Eight weeks later, plasma triglyceride (TG) and total cholesterol (TC) were determined. Sixteen animals with the highest levels of hyperlipidemia from the HFD group were randomly divided into two sub-groups: the untreated hyperlipidemia group (n=8) and the group treated with myakuryu (n=8). At the end of the sixteenth week, rheological and microcirculatory parameters were measured. Chemical analysis showed that myakuryu treatment caused significant reductions of plasma TG and TC levels (P

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod +/lacZ mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for β-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ -positive cells in the CNS were neurons. Large neurons, e.g. , mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.

A new technique is proposed to estimate the shear modulus (mu) and membrane surface viscosity (eta(m)) of red blood cell (RBC). Theoretical formulae for finding these two parameters are first derived based on the force balance on a RBC in a flow field of low viscosity. Different types of Ektacytometry are then used to measure relevant quantities. The obtained values (mu=6.1 x 10(-6)N/m, eta(m)=8.8 x10 (-7)Ns/m for normal RBC) are consistent with those previously found by micropipette technique and in AC electric field. The present technique is, however, much easier to operate and more advantageous in reflecting the average properties of a large quantity of RBCs, and it is much cheaper to be applied in clinical practice than any other method of measuring the two parameters. The sensitivity of the technique is demonstrated by testing RBCs treated with glutaraldehyde of different concentrations. This technique was demonstrated by the flow chamber.

The generation and progress of tumors are accompanied with a marked suppression of human immune system. To explore the mechanisms by which tumors escape from immune recognition, we studied the influences of tumor microenvironment on differentiation of dendritic cells (DCs), which play an important role in tumor immunology, by biophysical and immunological methods. It was found that the cytokines derived from tumors caused an increase in osmotic fragility and a decrease in membrane fluidity of DCs, disordering and elevated expression levels of cytoskeleton, and changes of the gene transcriptional levels and energy status of the cells. Moreover, IL-12 production and the expression levels of some surface-marker molecules were also suppressed. These changes led to impaired capabilities of antigen uptake, cell motility and naïve T cell activation; the abnormal biophysical characteristics of DCs may be one aspect of the immune escape mechanism of tumor. These results provide insights into the importance of the reconstruction of tumor microenvironment for immunotherapy based on the anti-cancer activities of DCs.

Hyperuricemia is a risk factor for various diseases, but knowledge on acute hyperuricemia is still not sufficient. The present study was aimed to investigate the effect of acute hyperuricemia on red blood cells from hemorheological point of view, and to provide the reference for clinical treatment. The rats were gavaged with 500 mg/kg hypoxanthine and intraperitoneally injected with 100 mg/kg oxonate to induce the model of acute hyperuricemia. The same volume of blood samples were drawn within time period of 0, 1, 2, 3 and 6 h, respectively, from the inner canthus of rats to measure the serum uric acid, hemorheological parameters and the malondialdehyde level. It was found that in each period of 1, 2 and 3 h, the rats had significantly higher levels of uric acid. The integrated deformation index and relax index were increased. The hemolysis rate was significantly reduced. The plasma malondialdehyde level was obviously decreased at the end of 2 h. The results suggested that short-term elevated uric acid could improve the hemorheological parameters and the lipid oxidative level in red blood cells.

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3'UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.

TRAIL can selectively induce rapid apoptosis of various types of tumor cells. We induced the expression of TRAIL in Jurkat cells, and measured the adhesion of cells to human umbilical vein endothelial cells (HUVECs) and laminin (LN) in a parallel plate flow chamber system and by using a colorimetric method. The apoptosis percentage, cycle distribution, intracellular Ca2+ concentration, and adhesion molecule expression of the cells were detected by flow cytometry. Cytoskeleton was observed with a laser confocal microscopy. The roles of adhesion molecules in the cell interaction was defined by their function blocking. The results showed that TRAIL attenuated the adhesion of Jurkat cells to HUVECs and LN, as well as their transendothelial migration. The increased apoptosis and G1-phase cell percentages, decreased intracellular Ca2+ concentration, depolymerized actin and impaired cell deformability could contribute to the decreased adhesion of Jurkat cells caused by TRAIL. Furthermore, CD11a was found to play a more important role than CD62L in the adhesion of Jurkat cells to HUVECs. These findings contribute to the knowledge on the role of TRAIL in tumor metastasis and provide mechanistic basis for the clinical application of TRAIL and tumor therapy. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc.

We investigated the biomechanical properties of reticulocytes obtained from an animal model of hemolytic anemia induced by antibody injection. The hemorheological indices, membrane viscoelasticity, membrane fluidity, and the secondary structure of membrane proteins of the reticulocytes were monitored continuously during the course of their maturation into erythrocytes. The results indicate that reticulocytes had lower deformability, lower membrane fluidity, greater viscoelastic modulus and lesser proportions of alpha-helices and beta-sheets in protein secondary structures than mature erythrocytes. All these indices approached to the level of normal erythrocytes when reticulocytes transformed during maturation. The results help to enhance our understanding of the biomechanical properties of the reticulocytes in their maturing process with clinical diagnosis significances.

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G0-G1 arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as α-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53. Cell Motil. Cytoskeleton 56:1–12, 2003. © 2003 Wiley-Liss, Inc.

Interlukin-10 (IL-10) is an immunomodulatory cytokine which predominantly induces immune-tolerance. It has been also identified as a major cytokine in the tumor microenvironment that markedly mediates tumor immune escape. Previous studies on the roles of IL-10 in tumor immunosuppression mainly focus on its biochemical effects. But the effects of IL-10 on the biophysical characteristics of immune cells are ill-defined. Dendritic cells (DCs) are the most potent antigen-presenting cells and play a key role in the anti-tumor immune response. IL-10 can affect the immune regulatory functions of DCs in various ways. In this study, we aim to explore the effects of IL-10 on the biophysical functions of mature DCs (mDCs). mDCs were treated with different concentrations of IL-10 and their biophysical characteristics were identified. The results showed that the biophysical properties of mDCs, including electrophoresis mobility, osmotic fragility and deformability, as well as their motilities, were impaired by IL-10. Meanwhile, the cytoskeleton (F-actin) of mDCs was reorganized by IL-10. IL-10 caused the alternations in the expressions of fasin1 and profilin1 as well as the phosphorylation of cofilin1 in a concentration-dependent fashion. Moreover, Fourier transformed infrared resonance data showed that IL-10 made the status of gene transcription and metabolic turnover of mDCs more active. These results demonstrate a new aspect of IL-10’s actions on the immune system and represent one of the mechanisms for immune escape of tumors. It may provide a valuable clue to optimize and improve the efficiency of DC-based immunotherapy against cancer.

Full-length erythrocyte tropomodulin (E-Tmod or Tmod1) isoform of 41 kDa is an actin nucleation protein and caps the pointed end of tropomyosin-coated actin filaments. It participates in the length control of short actin protofilaments in the erythrocyte membrane skeletal network as well as the organization of microfilaments in non-erythroid cells. Recently we discovered and characterized a truncated isoform of 29 kDa, which lacks the N-terminal sequence encoded by exons 1 and 2 required for nucleation and capping. Thus, it is important to study the expression pattern of solely the E-Tmod41 isoform in tissues. We utilized our exon 1 knockout (KO) mouse model with a knock-in lacZ reporter gene which reports the expression of E-Tmod41, but not E-Tmod29. Because this homozygous isoform-specific KO is an embryonic lethal mutation, we used heterozygous mice. X-gal staining localized specific signals at the single cell level and revealed a timed expression during embryonic development and restricted expression in adult mice. Our results showed that E-Tmod41 expressing cells include developing and young erythroid cells, developing somites, young fiber cells in the lens, certain subtype(s) of tubular cells in the kidney, smooth muscle cells in various tissues, and horizontal cells in the retina. A comparison with previous studies revealed that most if not all tissues known to express E-Tmod contained lacZ-expressing cells. Interestingly, some tubular cells were lacZ-positive while others in the same renal tubule were not, indicating heterogeneity within the tubular cells. Combined with double immunocytochemistry, we further localized E-Tmod41 to dendritic spines of horizontal cells. These timed and cell-type restricted expressions of E-Tmod41 suggest a role of actin nucleation and/or short actin protofilaments in these cell types and sub-cellular structures.