Sorafenib inhibits cAMP-dependent ERK activation, cell proliferation, and in vitro cyst growth of human ADPKD cyst epithelial cells

In autosomal dominant polycystic kidney disease (ADPKD), aberrant proliferation of the renal epithelial cells is responsible for the formation of numerable fluid-filled cysts, massively enlarged kidneys, and progressive loss of renal function. cAMP agonists, including arginine vasopressin, accelerate cyst epithelial cell proliferation through protein kinase A activation of the B-Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway. The mitogenic effect of cAMP is equally potent and additive to growth factor stimulation. Here, we determined whether Sorafenib (BAY 43–9006), a small molecule Raf inhibitor, inhibits proliferation of cells derived from the cysts of human ADPKD kidneys. We found that nanomolar concentrations of Sorafenib reduced the basal activity of ERK, inhibited cAMP-dependent activation of B-Raf and MEK/ERK signaling, and caused a concentration-dependent inhibition of cell proliferation induced by cAMP, epidermal growth factor, or the combination of the two agonists. Sorafenib completely blocked in vitro cyst growth of human ADPKD cystic cells cultured within a three-dimensional collagen gel. These data demonstrate that cAMP-dependent proliferation of human ADPKD cyst epithelial cells is blocked by Sorafenib and suggest that small molecule B-Raf inhibitors may be a therapeutic option to reduce the mitogenic effects of cAMP on cyst expansion.

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Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02188-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ryo Yokomizo ◽

Yukiko Fujiki ◽

Harue Kishigami ◽

Hiroshi Kishi ◽

Tohru Kiyono ◽

...

Keyword(s):

Epithelial Cells ◽

Stromal Cells ◽

Therapeutic Option ◽

Three Dimensional ◽

Fertility Treatment ◽

Feeder Cells ◽

Endometrial Stromal Cells ◽

Thin Endometrium ◽

Endometrial Epithelial Cells

Abstract Background Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro. Methods We prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells. Results Co-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model. Conclusions We herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

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Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

Odontology ◽

10.1007/s10266-021-00625-0 ◽

2021 ◽

Author(s):

Yoko Yamaguchi ◽

Akira Saito ◽

Masafumi Horie ◽

Akira Aoki ◽

Patrick Micke ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Experimental Studies ◽

Three Dimensional ◽

Collagen Gel ◽

Neutralizing Antibody ◽

Chronic Inflammatory Disease ◽

Collagen Degradation ◽

Hepatocyte Growth

AbstractPeriodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast–epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell–cell and cell–ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment.

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The tyrosine-kinase inhibitor Nintedanib ameliorates autosomal-dominant polycystic kidney disease

Cell Death and Disease ◽

10.1038/s41419-021-04248-9 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Abeda Jamadar ◽

Sreenath M. Suma ◽

Sijo Mathew ◽

Timothy A. Fields ◽

Darren P. Wallace ◽

...

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Kidney Disease ◽

Autosomal Dominant ◽

Renal Cyst ◽

Growth Factor Receptor ◽

Polycystic Kidney ◽

Autosomal Dominant Polycystic Kidney ◽

Cyst Growth

AbstractAutosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1f/fPkhd1Cre and Pkd1RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1f/fPkhd1Cre and Pkd1RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1RC/RC mice, but did not affect renal fibrosis in Pkd1f/fPkhd1Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.

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Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

The Journal of Cell Biology ◽

10.1083/jcb.95.1.333 ◽

1982 ◽

Vol 95 (1) ◽

pp. 333-339 ◽

Cited By ~ 364

Author(s):

G Greenburg ◽

E D Hay

Keyword(s):

Epithelial Cells ◽

Cell Polarity ◽

Three Dimensional ◽

Collagen Gel ◽

Mesenchymal Cells ◽

Apical Surface ◽

Collagen Gels ◽

Mesenchymal Transformation

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.

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Insulin and IGF-I induce pronounced hypertrophy of skeletal myofibers in tissue culture

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.3.c475 ◽

1991 ◽

Vol 260 (3) ◽

pp. C475-C484 ◽

Cited By ~ 142

Author(s):

H. H. Vandenburgh ◽

P. Karlisch ◽

J. Shansky ◽

R. Feldstein

Keyword(s):

Tissue Culture ◽

Growth Factor ◽

Three Dimensional ◽

Collagen Gel ◽

Insulin Like Growth Factor ◽

Cell Hyperplasia ◽

Factor Ii ◽

Igf I ◽

Collagen Gel Matrix

Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a defined serum-free medium for at least 6-7 days when embedded in a three-dimensional collagen gel matrix. Incubation of established myofiber cultures for 3-7 days with insulin (1 microM) or insulin-like growth factor I (IGF-I, 32 nM) stimulates both cell hyperplasia and myofiber hypertrophy. Mean myofiber diameter increases 71-98%. Insulin-like growth factor II stimulates cell hyperplasia but not myofiber hypertrophy. Cell growth results from a 42-62% increase in total protein synthesis and a 28-38% decrease in protein degradation. Myosin heavy-chain content increases 183-258% because of a 55% stimulation of myosin synthesis and 33-61% inhibition of degradation. Associated with myofiber hypertrophy is a 87-148% increase in the number of myofiber nuclei per unit myofiber length. The results indicate that insulin and IGF-I, but not IGF-II, can induce rapid myofiber hypertrophy in vitro, most likely by stimulating myoblast proliferation and/or fusion to established myofibers.

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Tolvaptan inhibits ERK-dependent cell proliferation, Cl− secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

AJP Renal Physiology ◽

10.1152/ajprenal.00243.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. F1005-F1013 ◽

Cited By ~ 87

Author(s):

Gail A. Reif ◽

Tamio Yamaguchi ◽

Emily Nivens ◽

Hiroyuki Fujiki ◽

Cibele S. Pinto ◽

...

Keyword(s):

Cell Proliferation ◽

Short Circuit ◽

Three Dimensional ◽

Short Circuit Current ◽

Collagen Matrix ◽

Fluid Secretion ◽

Intracellular Camp ◽

Erk Activity ◽

Cyst Growth

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl−-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl− secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10−9 M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC50 of ∼10−10 M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl− channel blocker CFTRinh-172, consistent with AVP-induced transepithelial Cl− secretion. Tolvaptan inhibited AVP-induced Cl− secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl−-dependent fluid secretion by human ADPKD cystic cells.

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VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/−) mice

AJP Cell Physiology ◽

10.1152/ajpcell.00038.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. C419-C428 ◽

Cited By ~ 41

Author(s):

Claudia R. Amura ◽

Kelley S. Brodsky ◽

Rachel Groff ◽

Vincent H. Gattone ◽

Norbert F. Voelkel ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Liver Cyst ◽

Cyst Fluid ◽

Vegf Receptor ◽

In Vitro Proliferation ◽

Vegf Signaling ◽

Cyst Growth ◽

Cyst Fluids

Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 ± 78, mouse 2,787 ± 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 ± 41, mouse 191 ± 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/−) liver cyst epithelial cells. Treatment of pkd2(WS25/−) liver cyst epithelial cells with VEGF (50–50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/−) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 ± 4.3%, SU-5416 1.8 ± 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 ± 0.2%, pkd2(WS25/−)-vehicle 10.8 ± 1.9%, pkd2(WS25/−)-SU-5416 4.8 ± 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/−) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/−) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease.

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Differential effects of retinoids on proliferation of bovine mammary epithelial cells in collagen gel culture

Journal of Dairy Research ◽

10.1017/s0022029901004782 ◽

2001 ◽

Vol 68 (2) ◽

pp. 157-164 ◽

Cited By ~ 16

Author(s):

STIG PURUP ◽

SØREN KROGH JENSEN ◽

KRIS SEJRSEN

Keyword(s):

Cell Proliferation ◽

Retinoic Acid ◽

Growth Factors ◽

Growth Factor ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Collagen Gel ◽

Mammary Epithelial ◽

Bovine Mammary Epithelial Cells ◽

Igf I

The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 μg/ml corresponded to a 75–95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 μg/ml) inhibited 68–85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-α), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml, retinol and retinoic acid inhibited respectively 24–38 and 44–52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10000 ng/ml, 61–71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-α was inhibited 42–64% by retinol and retinoic acid at 100 ng/ml, and 64–84% at 10000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.

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