Accepted Manuscript MicroRNAs in endothelial function: many challenges and early hopes for clinical applications Nikolaos G. Frangogiannis, MD PII:
S0735-1097(13)06083-X
DOI:
10.1016/j.jacc.2013.10.056
Reference:
JAC 19609
To appear in:
Journal of the American College of Cardiology
Received Date: 17 October 2013 Accepted Date: 23 October 2013
Please cite this article as: Frangogiannis NG, MicroRNAs in endothelial function: many challenges and early hopes for clinical applications, Journal of the American College of Cardiology (2013), doi: 10.1016/ j.jacc.2013.10.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
MicroRNAs in endothelial function: many challenges and early hopes for clinical applications
Nikolaos G Frangogiannis, MD
RI PT
Running Title: microRNAs in endothelial function
Address for correspondence: Nikolaos G Frangogiannis, MD
M AN U
Albert Einstein College of Medicine, Bronx NY.
SC
The Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology),
The Wilf Family Cardiovascular Research Institute
TE D
Albert Einstein College of Medicine
1300 Morris Park Avenue Forchheimer G46B Bronx NY 10461
Fax: 718-430-8989
EP
Tel: 718-430-3546
AC C
Email: [email protected]
Key Words: let-7g, endothelial function, TGF- β pathway, SIRT-1, PAI-1
No disclosures.
1
ACCEPTED MANUSCRIPT
The human genome contains over 20,000 protein-coding genes and at least as many noncoding RNA (ncRNA) genes, which are not translated into protein, but may contribute to regulation of gene expression by modulating transcriptional or post-transcriptional processes (1).
RI PT
Although they represent only a small fraction of the ncRNAs, the members of the evolutionarily ancient family of microRNAs (miRNAs) appear to play a uniquely important role in the pathogenesis of disease (2). Over the last five years, several members of the family have been
SC
identified as key modulators in a wide range of cardiac pathophysiologic conditions ranging from vascular disease (3) to heart failure and cardiac fibrosis (4).
M AN U
One of the best-studied and highly conserved miRNAs, the let-7 group consists of 12 members in mammals and corresponds to a C elegans counterpart that is an essential regulator of cell differentiation (5). Although a mounting body of evidence implicates let-7 transcripts in the pathobiology of cancer as tumor suppressors, their potential role in cardiovascular disease
TE D
remains unknown. In the current issue of the journal, Liao and co-workers (6) explore the role of Let-7g, one of the best-characterized members of the let-7 family, in endothelial function. Using in vitro experiments, animal investigations and samples from human patients, the authors found a
EP
protective effect of Let-7g on the endothelium, mediated through Transforming Growth Factor (TGF)- signaling. In vitro, Let-7g exerted anti-inflammatory actions on endothelial cells,
AC C
suppressed expression of Plasminogen Activator Inhibitor (PAI)-1 through downmodulation of TGF- signaling, and reduced endothelial cell senescence by increasing sirtuin 1 (SIRT1) expression. In vivo, injection of a Let-7g inhibitor in hyperlipidemic apolipoprotein E (ApoE) knockout mice increased vascular expression of TGF--inducible genes (including PAI-1), accentuated macrophage infiltration in the carotid artery and caused overgrowth of the intimamedia. In patients with lacunar stroke, low serum let-7g levels were associated with higher 2
ACCEPTED MANUSCRIPT
plasma PAI-1 levels. Taken together, the findings suggest that let-7g preserves endothelial function and suppresses inflammation induced by metabolic dysregulation through effects dependent, at least in part, through downmodulation of the TGF-/PAI-1 axis. Considering the
RI PT
recent explosion in studies implicating various miRNAs in phenotypic modulation of vascular cells, how does the current study impact our understanding of the pathophysiology of vascular disease?
SC
Let-7g as an endogenous inhibitor of endothelial inflammatory activation
Vascular homeostasis is dependent on preserved structure and optimal function of the
M AN U
endothelial layer (7). Normal healthy endothelial cells prevent leukocyte adhesion, inhibit thrombogenic processes and serve as an effective barrier between the blood and the vascular wall. In the presence of dyslipidemia,
pro-inflammatory activation of the endothelium by
oxidatively modified lipoproteins plays an important role in the pathogenesis of atherosclerosis
TE D
(8). In the presence of metabolic dysregulation or increased shear stress, a healthy endothelial layer may serve as a source of inhibitory mediators that prevent or restrain the inflammatory reaction. The current study suggests that let-7g may be a key endogenous suppressor of
EP
endothelial inflammatory activation; in its absence endothelial cells acquire a pro-inflammatory phenotype that triggers vascular injury and may also exert pro-thrombotic actions. Associative
AC C
evidence in both mice and human patients links hyperlipidemia with lower let-7g levels and suggests that downregulation of let-7g is associated with increased circulating levels of PAI-1 a marker of endothelial dysfunction and thrombogenic activation. Although the study reveals a novel and potentially important role for let-7g in the pathogenesis of vascular disease, the findings also raise several important questions:
3
ACCEPTED MANUSCRIPT
Is reduced let-7g expression causally involved in the pathogenesis of atherosclerotic disease? Although hyperlipidemia is associated with reduction in let-7g levels (9), it is unclear
RI PT
whether downmodulation of let-7g is a primary event that triggers inflammatory activation in subjects with metabolic dysregulation, or simply represents an epiphenomenon. The mechanisms of let-7g regulation in endothelial cells remain unknown. Growth factors and mitogen-activated
SC
protein kinase (MAPK) signaling have been demonstrated to reduce let-7g expression in carcinoma cells (10) and may have similar effects on the endothelium. A growing body of
M AN U
evidence suggests that inflammatory activation is associated with suppression of let-7g expression. In human volunteers, endotoxin infusion reduced leukocyte let-7g levels (11); however, whether innate immune signals are implicated in regulation of let-7g remains unknown. In tumor cells, let-7g loss increases invasiveness by accentuating matrix metalloproteinase
TE D
(MMP) activity (10). The involvement of MMPs in plaque rupture raises the intriguing possibility that let-7g downregulation in susceptible individuals may promote formation of unstable lesions.
EP
Which molecular signals mediate let-7g-induced preservation of endothelial function? The current investigation suggests the involvement of TGF- downmodulation in
AC C
mediating the effects of let-7g on the vasculature. This novel mechanistic finding is an important contribution of the study and, considering the broad involvement of the TGF- system in tissue injury, neoplasia and angiogenesis, may have implications in understanding the actions of let-7g in a wide range of pathophysiologic conditions. However, regulation of TGF- signaling is unlikely to be the only (or even the most important) action of Let-7g. Most miRNAs target multiple mRNAs; as a result, their actions are rarely explained through modulation of a single 4
ACCEPTED MANUSCRIPT
mediator. For example, the current study also demonstrates that let-7g downregulates expression of thrombospondin (TSP)-1, a matricellular protein with potent direct angiostatic properties (12), (13). Thus, the angiogenic effects of let-7g may be explained by the reduction of endothelial
RI PT
TSP-1 levels without the involvement of TGF- signaling. Moreover, interpretation of these in vitro observations is complicated by the notoriously pleiotropic and context-dependent actions of TGF-. Even the most robust and highly reproducible in vitro actions of TGF- are often of
SC
limited relevance for understanding pathophysiologic responses, because the in vivo context involves multiple cell types targeted by TGF-, and implicates many other mediators that may
M AN U
modulate its effects. Unfortunately, direct in vivo evidence demonstrating a critical role for TGF modulation in mediating effects of let-7g is lacking.
Are miRNAs therapeutic targets in vascular disease?
If Let-7g is required for suppression of endothelial inflammation under conditions of
TE D
metabolic dysregulation, mimicking let-7g actions may represent a promising therapy for atherosclerotic disease. Modulation of other miRNAs may also have therapeutic potential: systemic delivery of miRNA-181b inhibits vascular inflammation and attenuates atherosclerosis
EP
in ApoE KO mice (14), while miRNA-146 has been shown to inhibit endothelial proinflammatory activation (15). Despite these promising early observations, implementation of
AC C
miRNA targeting strategies in vascular disease remains a daunting task. In addition to the technical challenges in development of reproducible approaches for inhibition or repletion of selected miRNAs, concerns regarding the lack of specificity of miRNAs, their complex actions and the apparent redundancy of the miRNA system pose major challenges in therapeutic applications. Because most miRNAs target multiple pleiotropic mediators, the consequences of their modulation in human patients could be unpredictable. Moreover, because several distinct 5
ACCEPTED MANUSCRIPT
miRNAs can target the same gene, redundancy in their functions may complicate attempts for therapeutic modulation. These pitfalls are reflected in the conflicting findings often reported in the literature with the use of various approaches targeting specific miRNAs. In this rapidly
RI PT
evolving field, more robust in vivo experimentation and careful dissection of the pathophysiologic pathways is needed to support the potential clinical usefulness of miRNA-
AC C
EP
TE D
M AN U
SC
targeting strategies.
6
ACCEPTED MANUSCRIPT
REFERENCES: 1.
Lee TI, Young RA. Transcriptional regulation and its misregulation in disease. Cell
2.
RI PT
2013;152:1237-51. Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell 2012;148:1172-87.
Zampetaki A, Mayr M. MicroRNAs in vascular and metabolic disease. Circ Res
SC
3.
2012;110:508-22.
Kumarswamy R, Thum T. Non-coding RNAs in Cardiac Remodeling and Heart Failure. Circ Res 2013;113:676-89.
5.
Thornton JE, Gregory RI. How does Lin28 let-7 control development and disease? Trends Cell Biol 2012;22:474-82.
6.
M AN U
4.
Liao Y-C, Wang Y-S, Guo Y-C, Lin W-L, Chang M-H, Juo SH. Let-7g improves
Coll Cardiol 2013. 7.
TE D
multiple endothelial functions through targeting TGF-beta and SIRT-1 signaling. J Am
Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW. Endothelial-dependent
Libby P, Ridker PM, Hansson GK. Inflammation in atherosclerosis: from
AC C
8.
EP
mechanisms of leukocyte recruitment to the vascular wall. Circ Res 2007;101:234-47.
pathophysiology to practice. J Am Coll Cardiol 2009;54:2129-38. 9.
Chen KC, Hsieh IC, Hsi E, et al. Negative feedback regulation between microRNA let-7g and the oxLDL receptor LOX-1. J Cell Sci 2011;124:4115-24.
10.
Qian P, Zuo Z, Wu Z, et al. Pivotal role of reduced let-7g expression in breast cancer invasion and metastasis. Cancer Res 2011;71:6463-74.
7
ACCEPTED MANUSCRIPT
11.
Schmidt WM, Spiel AO, Jilma B, Wolzt M, Muller M. In vivo profile of the human leukocyte microRNA response to endotoxemia. Biochem Biophys Res Commun 2009;380:437-41. Frangogiannis NG. Matricellular proteins in cardiac adaptation and disease. Physiol Rev
RI PT
12.
2012;92:635-88. 13.
Gonzalez-Quesada C, Cavalera M, Biernacka A, et al. Thrombospondin-1 Induction in
SC
the Diabetic Myocardium Stabilizes the Cardiac Matrix, While Promoting Vascular
print). 14.
M AN U
Rarefaction Through Angiopoietin-2 Upregulation. Circ Res 2013 Sep 30 (epub ahead of
Sun X, He S, Wara AK, et al. Systemic Delivery of MicroRNA-181b Inhibits NF-kappaB Activation, Vascular Inflammation, and Atherosclerosis in Apoe-/- Mice. Circ Res 2013 Oct 1 (epub ahead of print).
TE D
Cheng HS, Sivachandran N, Lau A, et al. MicroRNA-146 represses endothelial activation
EP
by inhibiting pro-inflammatory pathways. EMBO Mol Med 2013;5:949-66.
AC C
15.
8
S0735-1097(13)06083-X
DOI:
10.1016/j.jacc.2013.10.056
Reference:
JAC 19609
To appear in:
Journal of the American College of Cardiology
Received Date: 17 October 2013 Accepted Date: 23 October 2013
Please cite this article as: Frangogiannis NG, MicroRNAs in endothelial function: many challenges and early hopes for clinical applications, Journal of the American College of Cardiology (2013), doi: 10.1016/ j.jacc.2013.10.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
MicroRNAs in endothelial function: many challenges and early hopes for clinical applications
Nikolaos G Frangogiannis, MD
RI PT
Running Title: microRNAs in endothelial function
Address for correspondence: Nikolaos G Frangogiannis, MD
M AN U
Albert Einstein College of Medicine, Bronx NY.
SC
The Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology),
The Wilf Family Cardiovascular Research Institute
TE D
Albert Einstein College of Medicine
1300 Morris Park Avenue Forchheimer G46B Bronx NY 10461
Fax: 718-430-8989
EP
Tel: 718-430-3546
AC C
Email: [email protected]
Key Words: let-7g, endothelial function, TGF- β pathway, SIRT-1, PAI-1
No disclosures.
1
ACCEPTED MANUSCRIPT
The human genome contains over 20,000 protein-coding genes and at least as many noncoding RNA (ncRNA) genes, which are not translated into protein, but may contribute to regulation of gene expression by modulating transcriptional or post-transcriptional processes (1).
RI PT
Although they represent only a small fraction of the ncRNAs, the members of the evolutionarily ancient family of microRNAs (miRNAs) appear to play a uniquely important role in the pathogenesis of disease (2). Over the last five years, several members of the family have been
SC
identified as key modulators in a wide range of cardiac pathophysiologic conditions ranging from vascular disease (3) to heart failure and cardiac fibrosis (4).
M AN U
One of the best-studied and highly conserved miRNAs, the let-7 group consists of 12 members in mammals and corresponds to a C elegans counterpart that is an essential regulator of cell differentiation (5). Although a mounting body of evidence implicates let-7 transcripts in the pathobiology of cancer as tumor suppressors, their potential role in cardiovascular disease
TE D
remains unknown. In the current issue of the journal, Liao and co-workers (6) explore the role of Let-7g, one of the best-characterized members of the let-7 family, in endothelial function. Using in vitro experiments, animal investigations and samples from human patients, the authors found a
EP
protective effect of Let-7g on the endothelium, mediated through Transforming Growth Factor (TGF)- signaling. In vitro, Let-7g exerted anti-inflammatory actions on endothelial cells,
AC C
suppressed expression of Plasminogen Activator Inhibitor (PAI)-1 through downmodulation of TGF- signaling, and reduced endothelial cell senescence by increasing sirtuin 1 (SIRT1) expression. In vivo, injection of a Let-7g inhibitor in hyperlipidemic apolipoprotein E (ApoE) knockout mice increased vascular expression of TGF--inducible genes (including PAI-1), accentuated macrophage infiltration in the carotid artery and caused overgrowth of the intimamedia. In patients with lacunar stroke, low serum let-7g levels were associated with higher 2
ACCEPTED MANUSCRIPT
plasma PAI-1 levels. Taken together, the findings suggest that let-7g preserves endothelial function and suppresses inflammation induced by metabolic dysregulation through effects dependent, at least in part, through downmodulation of the TGF-/PAI-1 axis. Considering the
RI PT
recent explosion in studies implicating various miRNAs in phenotypic modulation of vascular cells, how does the current study impact our understanding of the pathophysiology of vascular disease?
SC
Let-7g as an endogenous inhibitor of endothelial inflammatory activation
Vascular homeostasis is dependent on preserved structure and optimal function of the
M AN U
endothelial layer (7). Normal healthy endothelial cells prevent leukocyte adhesion, inhibit thrombogenic processes and serve as an effective barrier between the blood and the vascular wall. In the presence of dyslipidemia,
pro-inflammatory activation of the endothelium by
oxidatively modified lipoproteins plays an important role in the pathogenesis of atherosclerosis
TE D
(8). In the presence of metabolic dysregulation or increased shear stress, a healthy endothelial layer may serve as a source of inhibitory mediators that prevent or restrain the inflammatory reaction. The current study suggests that let-7g may be a key endogenous suppressor of
EP
endothelial inflammatory activation; in its absence endothelial cells acquire a pro-inflammatory phenotype that triggers vascular injury and may also exert pro-thrombotic actions. Associative
AC C
evidence in both mice and human patients links hyperlipidemia with lower let-7g levels and suggests that downregulation of let-7g is associated with increased circulating levels of PAI-1 a marker of endothelial dysfunction and thrombogenic activation. Although the study reveals a novel and potentially important role for let-7g in the pathogenesis of vascular disease, the findings also raise several important questions:
3
ACCEPTED MANUSCRIPT
Is reduced let-7g expression causally involved in the pathogenesis of atherosclerotic disease? Although hyperlipidemia is associated with reduction in let-7g levels (9), it is unclear
RI PT
whether downmodulation of let-7g is a primary event that triggers inflammatory activation in subjects with metabolic dysregulation, or simply represents an epiphenomenon. The mechanisms of let-7g regulation in endothelial cells remain unknown. Growth factors and mitogen-activated
SC
protein kinase (MAPK) signaling have been demonstrated to reduce let-7g expression in carcinoma cells (10) and may have similar effects on the endothelium. A growing body of
M AN U
evidence suggests that inflammatory activation is associated with suppression of let-7g expression. In human volunteers, endotoxin infusion reduced leukocyte let-7g levels (11); however, whether innate immune signals are implicated in regulation of let-7g remains unknown. In tumor cells, let-7g loss increases invasiveness by accentuating matrix metalloproteinase
TE D
(MMP) activity (10). The involvement of MMPs in plaque rupture raises the intriguing possibility that let-7g downregulation in susceptible individuals may promote formation of unstable lesions.
EP
Which molecular signals mediate let-7g-induced preservation of endothelial function? The current investigation suggests the involvement of TGF- downmodulation in
AC C
mediating the effects of let-7g on the vasculature. This novel mechanistic finding is an important contribution of the study and, considering the broad involvement of the TGF- system in tissue injury, neoplasia and angiogenesis, may have implications in understanding the actions of let-7g in a wide range of pathophysiologic conditions. However, regulation of TGF- signaling is unlikely to be the only (or even the most important) action of Let-7g. Most miRNAs target multiple mRNAs; as a result, their actions are rarely explained through modulation of a single 4
ACCEPTED MANUSCRIPT
mediator. For example, the current study also demonstrates that let-7g downregulates expression of thrombospondin (TSP)-1, a matricellular protein with potent direct angiostatic properties (12), (13). Thus, the angiogenic effects of let-7g may be explained by the reduction of endothelial
RI PT
TSP-1 levels without the involvement of TGF- signaling. Moreover, interpretation of these in vitro observations is complicated by the notoriously pleiotropic and context-dependent actions of TGF-. Even the most robust and highly reproducible in vitro actions of TGF- are often of
SC
limited relevance for understanding pathophysiologic responses, because the in vivo context involves multiple cell types targeted by TGF-, and implicates many other mediators that may
M AN U
modulate its effects. Unfortunately, direct in vivo evidence demonstrating a critical role for TGF modulation in mediating effects of let-7g is lacking.
Are miRNAs therapeutic targets in vascular disease?
If Let-7g is required for suppression of endothelial inflammation under conditions of
TE D
metabolic dysregulation, mimicking let-7g actions may represent a promising therapy for atherosclerotic disease. Modulation of other miRNAs may also have therapeutic potential: systemic delivery of miRNA-181b inhibits vascular inflammation and attenuates atherosclerosis
EP
in ApoE KO mice (14), while miRNA-146 has been shown to inhibit endothelial proinflammatory activation (15). Despite these promising early observations, implementation of
AC C
miRNA targeting strategies in vascular disease remains a daunting task. In addition to the technical challenges in development of reproducible approaches for inhibition or repletion of selected miRNAs, concerns regarding the lack of specificity of miRNAs, their complex actions and the apparent redundancy of the miRNA system pose major challenges in therapeutic applications. Because most miRNAs target multiple pleiotropic mediators, the consequences of their modulation in human patients could be unpredictable. Moreover, because several distinct 5
ACCEPTED MANUSCRIPT
miRNAs can target the same gene, redundancy in their functions may complicate attempts for therapeutic modulation. These pitfalls are reflected in the conflicting findings often reported in the literature with the use of various approaches targeting specific miRNAs. In this rapidly
RI PT
evolving field, more robust in vivo experimentation and careful dissection of the pathophysiologic pathways is needed to support the potential clinical usefulness of miRNA-
AC C
EP
TE D
M AN U
SC
targeting strategies.
6
ACCEPTED MANUSCRIPT
REFERENCES: 1.
Lee TI, Young RA. Transcriptional regulation and its misregulation in disease. Cell
2.
RI PT
2013;152:1237-51. Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell 2012;148:1172-87.
Zampetaki A, Mayr M. MicroRNAs in vascular and metabolic disease. Circ Res
SC
3.
2012;110:508-22.
Kumarswamy R, Thum T. Non-coding RNAs in Cardiac Remodeling and Heart Failure. Circ Res 2013;113:676-89.
5.
Thornton JE, Gregory RI. How does Lin28 let-7 control development and disease? Trends Cell Biol 2012;22:474-82.
6.
M AN U
4.
Liao Y-C, Wang Y-S, Guo Y-C, Lin W-L, Chang M-H, Juo SH. Let-7g improves
Coll Cardiol 2013. 7.
TE D
multiple endothelial functions through targeting TGF-beta and SIRT-1 signaling. J Am
Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW. Endothelial-dependent
Libby P, Ridker PM, Hansson GK. Inflammation in atherosclerosis: from
AC C
8.
EP
mechanisms of leukocyte recruitment to the vascular wall. Circ Res 2007;101:234-47.
pathophysiology to practice. J Am Coll Cardiol 2009;54:2129-38. 9.
Chen KC, Hsieh IC, Hsi E, et al. Negative feedback regulation between microRNA let-7g and the oxLDL receptor LOX-1. J Cell Sci 2011;124:4115-24.
10.
Qian P, Zuo Z, Wu Z, et al. Pivotal role of reduced let-7g expression in breast cancer invasion and metastasis. Cancer Res 2011;71:6463-74.
7
ACCEPTED MANUSCRIPT
11.
Schmidt WM, Spiel AO, Jilma B, Wolzt M, Muller M. In vivo profile of the human leukocyte microRNA response to endotoxemia. Biochem Biophys Res Commun 2009;380:437-41. Frangogiannis NG. Matricellular proteins in cardiac adaptation and disease. Physiol Rev
RI PT
12.
2012;92:635-88. 13.
Gonzalez-Quesada C, Cavalera M, Biernacka A, et al. Thrombospondin-1 Induction in
SC
the Diabetic Myocardium Stabilizes the Cardiac Matrix, While Promoting Vascular
print). 14.
M AN U
Rarefaction Through Angiopoietin-2 Upregulation. Circ Res 2013 Sep 30 (epub ahead of
Sun X, He S, Wara AK, et al. Systemic Delivery of MicroRNA-181b Inhibits NF-kappaB Activation, Vascular Inflammation, and Atherosclerosis in Apoe-/- Mice. Circ Res 2013 Oct 1 (epub ahead of print).
TE D
Cheng HS, Sivachandran N, Lau A, et al. MicroRNA-146 represses endothelial activation
EP
by inhibiting pro-inflammatory pathways. EMBO Mol Med 2013;5:949-66.
AC C
15.
8
