In vitro studies of intestinal fibrosis are confounded by spontaneous fibroblast activation on tissue culture polystyrene, hindering the investigation of early events tha... More
In vitro studies of intestinal fibrosis are confounded by spontaneous fibroblast activation on tissue culture polystyrene, hindering the investigation of early events that initiate fibrosis. Here, we present a statistically optimized culture protocol that suppresses fibroblast activation while preserving cell viability under standard culture conditions. Using a design of experiments (DOE) framework, we systematically evaluated combinations of extracellular matrix proteins and soluble factors to identify conditions that reduce myofibroblastic marker expression and extracellular matrix production. Fibroblasts cultured under optimized conditions remained spindle-shaped, exhibited low myofibroblastic marker expression, and showed reduced collagen and fibronectin secretion without evidence of cytotoxicity. The protocol was further validated in primary human colonic fibroblasts from both male and female donors, yielding consistent suppression of activation markers with high viability. This accessible and scalable approach provides a reproducible baseline for studying fibroblast activation and supports the use of DOE as a powerful strategy for defining microenvironmental conditions that regulate fibroblast behavior in vitro. Less
Cancer-associated fibroblasts (CAFs) transdifferentiated from hepatic stellate cells (HSCs) are a critical determinant of liver metastasis of colorectal cancer (CRC). How... More
Cancer-associated fibroblasts (CAFs) transdifferentiated from hepatic stellate cells (HSCs) are a critical determinant of liver metastasis of colorectal cancer (CRC). However, the mechanisms behind transforming growth factor β (TGF-β)-stimulated activation of HSCs into CAFs remain poorly understood. Immunoprecipitation coupled with mass spectrometry identified tuftelin 1 (TUFT1) as a novel TGF-β receptor II (TβRII) binding protein in primary human HSCs and immortalized LX2 cells. TUFT1 interacts with TβRII via its fragments (amino acids 1–86, 87–157), protecting TβRII from lysosomal degradation to facilitate TGF-β signaling and myofibroblastic activation of HSCs. Mechanistically, TUFT1 competes with caveolin-1 for TβRII binding, retrieving TβRII from the lipid rafts/caveolae-mediated degradation pathway and sorting it into the endosome-mediated trafficking and signaling pathway. Clinically, TUFT1 expression was confirmed in the CAFs of patient-derived colorectal cancer liver metastasis (CRCLM) tissues. Both protein and transcript analyses revealed higher TUFT1 expression in the CAFs of CRCLM than in HSCs. Furthermore, bulk RNA sequencing indicated that knocking down TUFT1 altered the TGF-β transcriptome of HSCs and suppressed HSC expression of tumor-promoting factors. In HSC/CRC co-implantation and portal vein tumor injection mouse models, targeting TUFT1 of HSCs inhibited HSC activation and restricted CRC growth in both subcutaneous and hepatic sites. Taken together, our findings uncover the novel function of TUFT1 in the hepatic tumor microenvironment, highlighting its role as a critical regulator of HSC activation and the pro-metastatic hepatic niche via promoting TβRII protein stability. Targeting TUFT1 in HSCs presents a promising therapeutic approach for combating CRCLM. Less
Background:
Myocardial fibrosis, characterized by excessive collagen deposition and fibroblast activation, is a pivotal pathological process driving heart failure after ... More
Background:
Myocardial fibrosis, characterized by excessive collagen deposition and fibroblast activation, is a pivotal pathological process driving heart failure after myocardial infarction (MI). Our prior research revealed that Brahma-related gene 1 (BRG1) expression is elevated after MI and exacerbated cardiac electrophysiological remodeling; however, its precise role and molecular mechanism in post-MI fibrosis remain undefined.
Methods:
BRG1 expression was assessed in a mouse MI model and in TGF-β1-stimulated cardiac fibroblasts (CFs). Gain- and loss-of-function studies were performed using adenoviral vectors, siRNA, and plasmids in vitro and in vivo. Cardiac function and fibrosis were evaluated by echocardiography and histology. The molecular mechanism was dissected through co-immunoprecipitation (Co-IP), dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), and functional rescue experiments targeting the PP2A/Smad3 axis.
Results:
BRG1 was upregulated in fibrotic mouse hearts post-MI and in activated CFs. In vivo, BRG1 knockdown via AAV9-shRNA improved cardiac function, reduced infarct size, and attenuated fibrosis. In vitro, BRG1 promoted CFs proliferation, migration, and collagen production. Mechanistically, TGF-β1 enhanced the interaction between BRG1 and the transcription factor ZEB1. This complex transcriptionally repressed Ppp2r1a, the gene encoding the PP2A structural subunit Aα, leading to diminished PP2A activity. Consequently, Smad3 phosphorylation and nuclear translocation were enhanced, amplifying the pro-fibrotic TGF-β/Smad3 cascade. Crucially, ZEB1 knockdown or PP2A inhibition (okadaic acid) could respectively block or rescue the fibrotic effects of BRG1. Finally, BRG1 knockdown similarly suppressed fibrotic activation in human CFs.
Conclusion:
Our study defines a novel BRG1/ZEB1/PP2A transcriptional axis as a key driver of myocardial fibrosis and suggests BRG1 as a potential therapeutic target for mitigating fibrotic remodeling after MI. Less
Hematogenous metastasis is the leading cause of cancer mortality, with dysfunction of pericytes, key components of tumor vessels, playing a central role in facilitating m... More
Hematogenous metastasis is the leading cause of cancer mortality, with dysfunction of pericytes, key components of tumor vessels, playing a central role in facilitating metastatic spread. Although anti-pericyte therapies are gaining recognition for treating metastasis, current strategies that directly eliminate tumor pericytes (TPCs) may increase vascular leakiness, which paradoxically promotes further metastasis. Here, we identify a TPC-specific transcription factor heterodimer, TCF21-TCF3, which drives metastasis by enhancing collagen hydroxylation and extracellular matrix deposition. Based on the TCF21 residues that interact with TCF3, we rationally design a peptide to disrupt their dimerization and downregulate TCF21-TCF3-dependent collagen deposition. Notably, in murine models of colorectal cancer and osteosarcoma, the TCF21-derived peptide significantly inhibits metastasis by restoring the physiological gatekeeper function of pericytes on vessels, offering a potential therapeutic strategy to target TPCs and suppress metastasis. Our findings reveal a TPC-specific transcription factor heterodimer and provide a promising pericyte-targeting strategy for preventing hematogenous metastasis. Less
Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is importa... More
Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2–induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2–induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2–induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells. Less
A mounting body of evidence in cancer research suggests that the local microenvironment of tumor cells has a profound influence on cancer progression and metastasis. In v... More
A mounting body of evidence in cancer research suggests that the local microenvironment of tumor cells has a profound influence on cancer progression and metastasis. In vitro studies on the tumor microenvironment and its pharmacological modulation, however, are often hampered by the technical challenges associated with creating physiological cell culture environments that integrate cancer cells with the key components of their native niche such as neighboring cells and extracellular matrix (ECM) to mimic complex microarchitecture of cancerous tissue. Using earlystage breast cancer as a model disease, here we describe a biomimetic microengineering strategy to reconstitute three-dimensional (3D) structural organization and microenvironment of breast tumors in human cell-based in vitro models. Specifically, we developed a microsystem that enabled co-culture of breast tumor spheroids with human mammary ductal epithelial cells and mammary fibroblasts in a compartmentalized 3D microfluidic device to replicate microarchitecture of breast ductal carcinoma in situ (DCIS). We also explored the potential of this breast cancer-on-a-chip system as a drug screening platform by evaluating the efficacy and toxicity of an anticancer drug (paclitaxel). Our microengineered disease model represents the first critical step towards recapitulating pathophysiological complexity of breast cancer, and may serve as an enabling tool to systematically examine the contribution of the breast cancer microenvironment to the progression of DCIS to an invasive form of the disease. Less
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy an... More
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples. Less
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy an... More
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples. Less
Treatment options for late stage prostate and colon cancer are limited and there is an urgent need to develop more effective and targeted novel therapies, which starts wi... More
Treatment options for late stage prostate and colon cancer are limited and there is an urgent need to develop more effective and targeted novel therapies, which starts with identification and validation of novel therapeutic targets. Recent clinical studies have demonstrated that tissue inhibitor matrix metalloproteinase-1 (TIMP-1) levels are elevated in cancer patient plasma and elevated TIMP-1 levels are associated with worse clinical outcomes. However, it is unknown whether TIMP-1 serves merely as a biomarker of cancer progression or has a functional role in promoting cancer progression and can serve as a cancer therapeutic target, which is the main objective of this study. Here, we show that stroma of human prostate and colon cancer express higher levels of TIMP-1 compared to their normal counterparts and increased expression of TIMP-1 promotes in vivo growth of both cancer types. We demonstrate for the first time that increased TIMP-1 expression stimulates accumulation of cancer associated fibroblasts (CAFs) within prostate and colon cancer tissues and that TIMP-1 enhances prostate CAF proliferation and migration in vitro and promotes ERK1/2 kinase activation in these CAF cells. Our results establish the novel promotive effects of TIMP-1 on cancer progression and on accumulation of CAFs that in turn provides a pro-tumor microenvironment. Together, these results establish the potential of TIMP-1 as a novel target for cancer therapy and the mechanism underlying the pro-tumor activity of TIMP-1. Less
Objective: Antibacterial primer and adhesive are promising to help combat biofilms and recurrent caries. The objectives of this study were to compare novel bonding agent ... More
Objective: Antibacterial primer and adhesive are promising to help combat biofilms and recurrent caries. The objectives of this study were to compare novel bonding agent containing quaternary ammonium dimethacrylate (QADM) with bonding agent containing nanoparticles of silver (NAg) in antibacterial activity, contact-inhibition vs. long-distance inhibition, glucosyltransferases (gtf) gene expressions, and cytotoxicity for the first time. Methods: QADM and NAg were incorporated into Scotchbond Multi-Purpose adhesive and primer. Microtensile dentin bond strength was measured. Streptococcus mutans (S. mutans) biofilm on resin surface (contact-inhibition) as well as S. mutans in culture medium away from the resin surface (long-distance inhibition) were tested for metabolic activity, colony-forming units (CFUs), lactic acid production, and gtf gene expressions. Eluents from cured primer/adhesive samples were used to examine cytotoxicity against human gingival fibroblasts. Results: Bonding agent with QADM greatly reduced CFU and lactic acid of biofilms on the resin surface (p<0.05), while having no effect on S. mutans in culture medium away from the resin surface. In contrast, bonding agent with NAg inhibited not only S. mutans on the resin surface, but also S. mutans in culture medium away from the resin surface. Bonding agent with QADM suppressed gtfB, gtfC and gtfD gene expressions of S. mutans on its surface, but not away from its surface. Bonding agent with NAg suppressed S. mutans gene expressions both on its surface and away from its surface. Bonding agents with QADM and NAg did not adversely affect microtensile bond strength or fibroblast cytotoxicity, compared to control (p>0.1). Significance: QADM-containing adhesive had contact-inhibition and inhibited bacteria on its surface, but not away from its surface. NAg-containing adhesive had long-distance killing capability and inhibited bacteria on its surface and away from its surface. The novel antibacterial adhesives are promising for caries-inhibition restorations, and QADM and NAg could be complimentary agents in inhibiting bacteria on resin surface as well as away from resin surface. Published by Elsevier Ltd. Less
Treatment options for late stage prostate and colon cancer are limited and there is an urgent need to develop more effective and targeted novel therapies, which starts wi... More
Treatment options for late stage prostate and colon cancer are limited and there is an urgent need to develop more effective and targeted novel therapies, which starts with identification and validation of novel therapeutic targets. Recent clinical studies have demonstrated that tissue inhibitor matrix metalloproteinase-1 (TIMP-1) levels are elevated in cancer patient plasma and elevated TIMP-1 levels are associated with worse clinical outcomes. However, it is unknown whether TIMP-1 serves merely as a biomarker of cancer progression or has a functional role in promoting cancer progression and can serve as a cancer therapeutic target, which is the main objective of this study. Here, we show that stroma of human prostate and colon cancer express higher levels of TIMP-1 compared to their normal counterparts and increased expression of TIMP-1 promotes in vivo growth of both cancer types. We demonstrate for the first time that increased TIMP-1 expression stimulates accumulation of cancer associated fibroblasts (CAFs) within prostate and colon cancer tissues and that TIMP-1 enhances prostate CAF proliferation and migration in vitro and promotes ERK1/2 kinase activation in these CAF cells. Our results establish the novel promotive effects of TIMP-1 on cancer progression and on accumulation of CAFs that in turn provides a pro-tumor microenvironment. Together, these results establish the potential of TIMP-1 as a novel target for cancer therapy and the mechanism underlying the pro-tumor activity of TIMP-1. Less
Purpose: To determine the ocular anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist (SEGRA) in primary human conjunctival fibroblasts ... More
Purpose: To determine the ocular anti-allergic effects of mapracorat, a novel selective glucocorticoid receptor agonist (SEGRA) in primary human conjunctival fibroblasts and epithelial cells. Less
Objectives: The objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium... More
Objectives: The objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and nanoparticles of silver (NAg), on dentine bond strength, dental plaque microcosm biofilm response, and fibroblast cytotoxicity for the first time. Methods: Scotchbond Multi-Purpose (SBMP) was used as the parent bonding agent. Four primers were tested: SBMP primer control (referred to as "P"), P+5% MDPB, P+0.05% NAg, and P+5% MDPB+0.05% NAg. Dentine shear bond strengths were measured using extracted human teeth. Biofilms from the mixed saliva of 10 donors were cultured to investigate metabolic activity, colony-forming units (CFU), and lactic acid production. Human fibroblast cytotoxicity of the four primers was tested in vitro. Results: Incorporating MDPB and NAg into primer did not reduce dentine bond strength compared to control (p>0.1). SEM revealed well-bonded adhesive-dentine interfaces with numerous resin tags. MDPB or NAg each greatly reduced biofilm viability and acid production, compared to control. Dual agents MDPB+NAg had a much stronger effect than either agent alone (p<0.05), increasing inhibition zone size and reducing metabolic activity, CFU and lactic acid by an order of magnitude, compared to control. There was no difference in cytotoxicity between commercial control and antibacterial primers (p>0.1). Conclusions: The method of using dual agents MDPB+NAg in the primer yielded potent antibacterial properties. Hence, this method may be promising to combat residual bacteria in tooth cavity and invading bacteria at the margins. The dual agents MDPB+NAg may have wide applicability to other adhesives, composites, sealants and cements to inhibit biofilms and caries. Less
Background: Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit.... More
Background: Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit. Because EC do not burn tobacco, they may be safer than conventional cigarettes. Their use is rapidly increasing worldwide with little prior testing of their aerosol.
Objectives: We tested the hypothesis that EC aerosol contains metals derived from various components in EC.
Methods: Cartomizer contents and aerosols were analyzed using light and electron microscopy, cytotoxicity testing, x-ray microanalysis, particle counting, and inductively coupled plasma optical emission spectrometry.
Results: The filament, a nickel-chromium wire, was coupled to a thicker copper wire coated with silver. The silver coating was sometimes missing. Four tin solder joints attached the wires to each other and coupled the copper/silver wire to the air tube and mouthpiece. All cartomizers had evidence of use before packaging (burn spots on the fibers and electrophoretic movement of fluid in the fibers). Fibers in two cartomizers had green deposits that contained copper. Centrifugation of the fibers produced large pellets containing tin. Tin particles and tin whiskers were identified in cartridge fluid and outer fibers. Cartomizer fluid with tin particles was cytotoxic in assays using human pulmonary fibroblasts. The aerosol contained particles >1 µm comprised of tin, silver, iron, nickel, aluminum, and silicate and nanoparticles (<100 nm) of tin, chromium and nickel. The concentrations of nine of eleven elements in EC aerosol were higher than or equal to the corresponding concentrations in conventional cigarette smoke. Many of the elements identified in EC aerosol are known to cause respiratory distress and disease.
Conclusions: The presence of metal and silicate particles in cartomizer aerosol demonstrates the need for improved quality control in EC design and manufacture and studies on how EC aerosol impacts the health of users and bystanders. Less
Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B-type natriuretic peptide ... More
Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B-type natriuretic peptide (BNP) is anti-fibrotic, inhibits collagen production, augments matrix metalloproteinases, and suppresses CF proliferation. Recently, we demonstrated that the ECM protein fibronectin (FN) augmented production of BNP's second messenger, 3', 5' cyclic guanosine monophosphate (cGMP) in CFs, supporting crosstalk between FN, BNP, and its receptor, natriuretic peptide receptor A (NPR-A). Here, we address the specificity of FN to augment cGMP generation by investigating other matrix proteins, including collagen IV which contains RGD motifs and collagen I and poly-L-lysine, which have no RGD domain. Collagen IV showed increased cGMP generation to BNP similar to FN. Collagen I and poly-L-lysine had no effect. As FN also interacts with integrins, we then examined the effect of integrin receptor antibody blockade on BNP-mediated cGMP production. On FN plates, antibodies blocking RGD-binding domains of several integrin subtypes had little effect, while a non-RGD domain interfering integrin alphavbeta3 antibody augmented cGMP production. Further, on uncoated plates, integrin alphavbeta3 blockade continued to potentiate the BNP/cGMP response. These studies suggest that both RGD containing ECM proteins and integrins may interact with BNP/NPR-A to modulate cGMP generation. Less
The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questi... More
The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questions about how this regulation functions and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication. We report here that after serum deprivation several human cell lines tested promptly export a substantial amount of miRNAs into the culture medium and the export process is largely energy dependent. The exported miRNAs are found both within and outside of the 16.5 and 120 K centrifugation pellets which contain most of the known cell-derived vesicles, the microvesicles and exosomes. We have identified some candidate proteins involved in this system, and one of these proteins may also play a role in protecting extracellular miRNAs from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that is consistent with the cell-cell communication hypothesis. Less
Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B-type natriuretic peptide ... More
Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B-type natriuretic peptide (BNP) is anti-fibrotic, inhibits collagen production, augments matrix metalloproteinases, and suppresses CF proliferation. Recently, we demonstrated that the ECM protein fibronectin (FN) augmented production of BNP's second messenger, 3', 5' cyclic guanosine monophosphate (cGMP) in CFs, supporting crosstalk between FN, BNP, and its receptor, natriuretic peptide receptor A (NPR-A). Here, we address the specificity of FN to augment cGMP generation by investigating other matrix proteins, including collagen IV which contains RGD motifs and collagen I and poly-L-lysine, which have no RGD domain. Collagen IV showed increased cGMP generation to BNP similar to FN. Collagen I and poly-L-lysine had no effect. As FN also interacts with integrins, we then examined the effect of integrin receptor antibody blockade on BNP-mediated cGMP production. On FN plates, antibodies blocking RGD-binding domains of several integrin subtypes had little effect, while a non-RGD domain interfering integrin alphavbeta3 antibody augmented cGMP production. Further, on uncoated plates, integrin alphavbeta3 blockade continued to potentiate the BNP/cGMP response. These studies suggest that both RGD containing ECM proteins and integrins may interact with BNP/NPR-A to modulate cGMP generation. Less
The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questi... More
The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questions about how this regulation functions and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell-cell communication. We report here that after serum deprivation several human cell lines tested promptly export a substantial amount of miRNAs into the culture medium and the export process is largely energy dependent. The exported miRNAs are found both within and outside of the 16.5 and 120 K centrifugation pellets which contain most of the known cell-derived vesicles, the microvesicles and exosomes. We have identified some candidate proteins involved in this system, and one of these proteins may also play a role in protecting extracellular miRNAs from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that is consistent with the cell-cell communication hypothesis. Less
