Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • General Statistics
    • Editorial Board
    • Editors
    • Information for Advertisers
    • Author Reprints
    • Commercial Reprints
    • Customer Service and Ordering Information
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • AHA Guidelines and Statements
    • Acknowledgment of Reviewers
    • Clinical Implications
    • Clinical-Pathological Conferences
    • Controversies in Hypertension
    • Editors' Picks
    • Guidelines Debate
    • Meeting Abstracts
    • Recent Advances in Hypertension
    • SPRINT Trial: the Conversation Continues
  • Resources
    • Instructions to Reviewers
    • Instructions for Authors
    • →Article Types
    • → Submission Guidelines
      • Research Guidelines
        • Minimum Information About Microarray Data Experiments (MIAME)
      • Abstract
      • Acknowledgments
      • Clinical Implications (Only by invitation)
      • Conflict(s) of Interest/Disclosure(s) Statement
      • Figure Legends
      • Figures
      • Novelty and Significance: 1) What Is New, 2) What Is Relevant?
      • References
      • Sources of Funding
      • Tables
      • Text
      • Title Page
      • Online/Data Supplement
    • →Tips for Easier Manuscript Submission
    • → General Instructions for Revised Manuscripts
      • Change of Authorship Form
    • → Costs to Authors
    • → Open Access, Repositories, & Author Rights Q&A
    • Permissions to Reprint Figures and Tables
    • Journal Policies
    • Scientific Councils
    • AHA Journals RSS Feeds
    • International Users
    • AHA Newsroom
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Facebook
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Hypertension

  • My alerts
  • Sign In
  • Join

  • Facebook
  • Twitter
  • Home
  • About this Journal
    • General Statistics
    • Editorial Board
    • Editors
    • Information for Advertisers
    • Author Reprints
    • Commercial Reprints
    • Customer Service and Ordering Information
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • AHA Guidelines and Statements
    • Acknowledgment of Reviewers
    • Clinical Implications
    • Clinical-Pathological Conferences
    • Controversies in Hypertension
    • Editors' Picks
    • Guidelines Debate
    • Meeting Abstracts
    • Recent Advances in Hypertension
    • SPRINT Trial: the Conversation Continues
  • Resources
    • Instructions to Reviewers
    • Instructions for Authors
    • →Article Types
    • → Submission Guidelines
    • →Tips for Easier Manuscript Submission
    • → General Instructions for Revised Manuscripts
    • → Costs to Authors
    • → Open Access, Repositories, & Author Rights Q&A
    • Permissions to Reprint Figures and Tables
    • Journal Policies
    • Scientific Councils
    • AHA Journals RSS Feeds
    • International Users
    • AHA Newsroom
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
Cold-Induced Hypertension

Ribonucleic Acid Interference Knockdown of Interleukin 6 Attenuates Cold-Induced Hypertension

Patrick Crosswhite, Zhongjie Sun
Download PDF
https://doi.org/10.1161/HYPERTENSIONAHA.109.146902
Hypertension. 2010;55:1484-1491
Originally published May 19, 2010
Patrick Crosswhite
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Zhongjie Sun
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics
  • eLetters

Jump to

  • Article
    • Abstract
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • References
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics
  • eLetters
Loading

Abstract

The purpose of this study was to determine the role of the proinflammatory cytokine interleukin (IL) 6 in cold-induced hypertension. Four groups of male Sprague-Dawley rats were used (6 rats per group). After blood pressure was stabilized, 3 groups received intravenous delivery of adenoassociated virus carrying IL-6 small hairpin RNA (shRNA), adenoassociated virus carrying scrambled shRNA, and PBS, respectively, before exposure to a cold environment (5°C). The last group received PBS and was kept at room temperature (25°C, warm) as a control. Adenoassociated virus delivery of IL-6 shRNA significantly attenuated cold-induced elevation of systolic blood pressure and kept it at the control level for ≤7 weeks (length of the study). Chronic exposure to cold upregulated IL-6 expression in aorta, heart, and kidneys and increased macrophage and T-cell infiltration in kidneys, suggesting that cold exposure increases inflammation. IL-6 shRNA delivery abolished the cold-induced upregulation of IL-6, indicating effective silence of IL-6. Interestingly, RNA interference knockdown of IL-6 prevented cold-induced inflammation, as evidenced by a complete inhibition of tumor necrosis factor-α expression and leukocyte infiltration by IL-6 shRNA. RNA interference knockdown of IL-6 significantly decreased the cold-induced increase in vascular superoxide production. It is noted that IL-6 shRNA abolished the cold-induced increase in collagen deposition in the heart, suggesting that inflammation is involved in cold-induced cardiac remodeling. Cold exposure caused glomerular collapses, which could be prevented by knockdown of IL-6, suggesting an important role of inflammation in cold-induced renal damage. In conclusion, cold exposure increased IL-6 expression and inflammation, which play critical roles in the pathogenesis of cold-induced hypertension and cardiac and renal damage.

  • cold exposure
  • inflammation
  • interleukin 6
  • short-hairpin siRNA
  • RNAi
  • adenoassociated virus

It is well documented that cold temperatures have adverse effects on the human cardiovascular system.1 The prevalence of hypertension and related cardiovascular disease is higher in people who live in colder climates. The mortality and morbidity of cardiovascular diseases peak during the winter months in the United States.2–4 In addition, cold temperatures exacerbate hypertension in hypertensive patients.5–7 Therefore, it is important to fully understand the mechanism mediating cold-induced elevation of blood pressure (BP).

Cold-induced hypertension (CIH) represents an excellent model for studying environmentally induced hypertension. CIH is a “naturally occurring” form of experimental hypertension that requires no genetic manipulation, surgical intervention, or excessive drug or hormone administration.1,8–10 Previous studies have provided a central role for the sympathetic nervous system in initiating CIH via activation of the renin-angiotensin system.1,11,12 Activation of the renin-angiotensin system results in increased levels of angiotensin II and aldosterone. Other than its potent vasoactive effects, angiotensin II has been demonstrated to influence many inflammatory processes.13,14 Excessive aldosterone levels have also been shown to increase inflammation.15

Located in the short arm of chromosome 7 (7p21) in humans, interleukin (IL) 6 is a pleiotropic cytokine with multiple biological roles in many different types of cell.16 The functions of IL-6 include induction of both local and systemic inflammatory responses, regulation of immune reaction, and hematopoiesis.17 In addition, IL-6 also induces proliferation and differentiation of T cells, as well as terminal differentiation of autoantibody-producing B cells.17 An overproduction of IL-6 thus exacerbates the immune reaction. Also, circulating IL-6 has been linked to central obesity, hypertension, and insulin resistance.16 In men, the proinflammatory cytokine IL-6 has been shown to be associated with elevated BP.18 Several reports have shown increased plasma levels of IL-6 in hypertensive patients.19–21 In addition, infusion of IL-6 into pregnant female rats induces preeclampsia (pregnancy-induced hypertension).22 Furthermore, angiotensin receptor blockers have been reported to reduce inflammatory mediator levels in hypertensive patients, specifically IL-6 and tumor necrosis factor (TNF)-α levels.15

Although the association of IL-6 and hypertension has been reported, the cause-and-effect relationship is not clear. The purpose of this study was to examine the role of IL-6 in the development of hypertension using the CIH model. We hypothesized that in vivo knockdown of IL-6 by RNA interference (RNAi) silencing would decrease inflammatory infiltrates and attenuate cold-induced elevation of BP.

Methods

For a full description of the Materials and Methods, please see the online Data Supplement at http://hyper.ahajournals.org.

Adenoassociated Virus IL-6 Generation

The procedure for constructing the recombinant adenoassociated virus (AAV) 2 carrying the rat IL-6 small hairpin (sh)RNA sequence was described in detail in the online Data Supplement.

Additional AAV Generation

AAV carrying Scrambled shRNA (ScrshRNA) was constructed and used as a control construct. Scrambled shRNA has been confirmed by BD Biosciences not to match any known gene sequence.

Packaging of Recombinant Plasmids of AAV With IL-6-shRNA

AAV.IL-6 and AAV.ScrshRNA were packaged with pHelper and pAAV-Helper to produce recombinant viruses as described in our recent studies.23,24 For the virus packaging procedure, please see the online Data Supplement.

Animal Study Protocols

This study was carried out according to the guidelines of the National Institutes of Health on the Care and Use of Laboratory Animals. This project was approved by the Institutional Animal Care and Use Committee. Four groups of male Sprague-Dawley rats (145 to 180 g, 6 rats per group) were allowed to acclimate for a week. After acclimation, resting systolic BP was measured twice weekly at room temperature from the tail of each unanesthetized rat by using a tail-cuff method with slight warming (28°C) but not heating of the tail using a CODA 6 BP Monitoring System (Kent Scientific). The volume-based tail-cuff measurements of BP have been validated by using a telemetry system.25 After 2 stable BP readings were obtained, the 4 groups of rats received a single injection via the tail vein of AAV.IL-6 shRNA, AAV.ScrshRNA, PBS, and PBS, respectively. The AAV complexes were delivered at 1.2×108 plaque-forming units per rat (0.5 mL). After injection, 3 groups (AAV.IL-6 shRNA, AAV.ScrshRNA, and 1 PBS) were moved into to a climate-controlled walk-in chamber (5±2°C), whereas the remaining PBS group was kept in an identical chamber maintained at room temperature (25±2°C, warm). BP and body weight were measured at least once per week until week 8 postinjection, when all of the animals were euthanized. For detailed procedures, please see the online Data Supplement.

Western Blot Analysis of IL-6 and TNF-α Protein Expressions in Tissue

IL-6 and TNF-α protein expressions in kidneys and arteries were measured using Western blot, as described in our previous studies.23,24 For details, please see the online Data Supplement.

Immunohistochemical Analysis of IL-6 Expression and Macrophage and T-Cell Infiltration

IL-6 expression and macrophage and T-cell infiltration were analyzed using IL-6 antibody and CD-3 and CD-68 markers, respectively. For details, please see the online Data Supplement.

Measurement of In Situ Vascular Superoxide Production

The in situ superoxide production was measured in aortas using the oxidation-sensitive dye dihydroethidium (DHE). For details, please see the online Data Supplement.

Statistical Analysis

The data for BP and body weight were analyzed by a repeated-measures 1-way ANOVA. The protein expression and reactive oxygen species levels were analyzed by 1-way ANOVA. Tukey multiple comparison tests were used to assess the significance of differences between means. Significance was set at a 95% confidence limit.

Results

IL-6 shRNA Delivery Attenuated the Cold-Induced BP Increase

IL-6 shRNA significantly decreased cold-induced elevation of systolic BP compared with the PBS cold and ScrshRNA cold groups (Figure 1). RNAi knockdown of IL-6 maintained BPs at the level of the PBS warm group (control). It is noted that one single dose of AAV.IL-6 shRNA controlled hypertension for ≥7 weeks (length of the study; Figure 1). Body weight was not significantly different between groups both before and after the single injection of viral complexes (Figure S2, available in the online Data Supplement), suggesting that AAV-2 had no adverse effects on body weight gains.

Figure1
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 1. IL-6 shRNA delivery attenuated cold-induced elevation of blood pressure. Intravenous injections of AAV.ScrshRNA, AAV.IL-6 shRNA, and PBS were administered before exposure to cold. Systolic BP was measured weekly. Data=mean±SE; +P<0.05, +++P<0.001 vs the ScrshRNA cold. n=6.

IL-6 shRNA Delivery Decreased Cold-Induced Increases in IL-6 Expression in the Kidney

The PBS cold group showed a significant increase in IL-6 protein expression compared with the PBS warm group (Figure 2A and 2B), suggesting that cold exposure may increase inflammation. IL-6 shRNA significantly decreased the cold-induced increase in IL-6 expression versus the ScrshRNA cold group and the PBS cold group (Figure 2A and 2B). The immunohistochemical (IHC) analysis revealed that cold exposure increased IL-6 expression (brown staining) around the glomeruli and tubules in the ScrshRNA Cold group and the PBS Cold group versus the PBS Warm group (Figure 2C and 2D). IL-6 expression was decreased significantly in the IL-6shRNA Cold group (Figure 2C and 2D). These results indicate that IL-6 gene was effectively silenced by IL-6shRNA.

Figure2
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 2. IL-6 shRNA delivery decreased IL-6 expression in the kidney. Western blot analysis of IL-6 protein expression in kidney homogenates (A and B). IHC analysis of IL-6 expression in the kidney (C and D). Arrows indicate IL-6 expression (brown staining). *P<0.05, **P<0.01, ***P<0.001 vs the PBS warm group; ++P<0.01, +++P<0.001 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

IL-6shRNA Delivery Attenuated Cold-Induced Macrophage and T-Cell Infiltration in the Kidney

The IHC analysis showed that CD-68 staining and the number of CD-68+ cells were increased significantly in kidneys in PBS cold and ScrshRNA cold groups (Figure 3A through 3C), indicating that cold exposure increased macrophage infiltration. Macrophages infiltrated in the renal tubules and glomeruli (Figure 3C). IL-6 shRNA abolished the cold-induced increase in macrophage infiltration (Figure 3A through 3C). Cold exposure also increased T-cell (CD-3+ cell) infiltration in kidneys, which could be abolished by IL-6 shRNA (Figure 3D through 3F). T cells infiltrated in the renal tubules (Figure 3F). These results suggest that that silence of IL-6 prevented cold-induced inflammation.

Figure3
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 3. IL-6 shRNA delivery attenuated macrophage and T-cell infiltration in the kidney. Semiquantitative analysis of macrophage infiltration in the kidney, as measured by the density of CD-68 expression (A) and the average count of CD-68+ cells (B). The IHC analysis of macrophage infiltration in the kidney as measured by CD-68 staining (C). Semiquantitative analysis of T-cell infiltration in the kidney as measured by the density of CD-3 expression (D) and the average count of CD-3+ cells (E). The IHC analysis of T-cell infiltration in the kidney was measured by CD-3 staining (F). Arrows indicate positive staining of CD-68 or CD-3. Photos were taken at ×200. Scale bars indicate 100 μm. *P<0.05, **P<0.01, ***P<0.001 vs the PBS Warm group; +P<0.05, ++P<0.01, +++P<0.001 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

IL-6 shRNA Delivery Attenuated Cold-Induced Kidney Damage

Figure 4 showed glomerular collapses in the ScrshRNA cold and PBS cold groups, indicating that chronic exposure to cold caused structural damage in kidneys. RNAi knockdown of IL-6 abolished cold-induced glomerular collapses (Figure 4A and 4B).

Figure4
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 4. IL-6 shRNA delivery prevents cold-induced kidney damage. A, Hematoxylin/eosin staining in the kidney showing collapsed glomeruli. B, Quantitative analysis of glomerular collapse. Photos were taken at ×400. Scale bars represent 50 μm. *P<0.05, **P<0.01<0.001 vs the PBS warm group; ++P<0.01, +++P<0.001 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

IL-6 shRNA Delivery Abolished the Cold-Induced Increase in IL-6 and TNF-α Expression in Aorta

Western blot analysis showed significant increases in IL-6 and TNF-α protein expression in aortas in the ScshRNA cold and PBS cold groups compared with the PBS warm group (Figure 5), indicating that cold exposure may increase inflammation in the aorta. IL-6 shRNA delivery abolished the cold-induced increases in IL-6 and TNF-α in aortas (Figure 5). This result suggests that IL-6 may mediate the cold-induced increase in vascular TNF-α.

Figure5
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 5. IL-6 shRNA delivery decreased IL-6 and TNF-α expression in aorta. Quantitative analysis of IL-6 protein expression in abdominal aorta (A) and the representative Western blot bands of IL-6 (B). Quantitative analysis of TNF-α protein expression in abdominal aorta (C) and the representative Western blot bands of TNF-α (D). *P<0.05, **P<0.01 vs PBS warm group; +P<0.05, ++P<0.01 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

IL-6 shRNA Delivery Abolished the Cold-Induced Increase in Vascular Superoxide Production

In situ vascular superoxide production was evaluated using DHE staining. Cold exposure significantly increased superoxide production in aortas (Figure 6). RNAi silencing of IL-6 abolished the cold-induced increase in vascular superoxide production.

Figure6
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 6. IL-6 shRNA delivery decreased in situ superoxide production in the abdominal aorta. Quantification of superoxide staining in aortas (A). Sections of aortas were incubated with DHE, and red fluorescence was viewed using tetramethylrhodamine B isothiocyanate. 4′,6-Diamidino-2-phenylindole (DAPI) was used to view nuclear staining. Aortic segments showing DHE staining (red), DAPI staining (blue), and a merge of DHE and DAPI, respectively (B). Sections were viewed at ×200. **P<0.01 vs the PBS warm group; ++P<0.01, +++P<0.001 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

IL-6 shRNA Delivery Abolished the Cold-Induced Increases in IL-6 Expression in the Heart and Prevented Collagen Deposition Around the Coronary Arteries

IHC analysis showed a significant increase in cardiac IL-6 expression in the PBS cold and ScrshRNA cold groups (Figure 7A and 7B), suggesting that cold exposure increased inflammation in the heart. IL-6 shRNA significantly decreased IL-6 expression compared with the PBS cold and ScrshRNA cold groups (Figure 7A), confirming effective silencing of the IL-6 gene. A significant decrease in collagen deposition (blue staining) around the coronary arteries of the IL-6 shRNA cold group was observed compared with the PBS cold and ScrshRNA cold groups (Figure 7C and 7D), suggesting that RNAi silencing of IL-6 prevented heart remodeling. IL-6 shRNA decreased the cold-induced increase in heart weight but did not reduce it to the control level (Figure S1), suggesting partial attenuation of cold-induced cardiac hypertrophy.

Figure7
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 7. IL-6 shRNA delivery reduced IL-6 expression in the heart and prevented cardiac collagen deposition. Semiquantitative analysis of IL-6 expression in the heart (A) and the IHC analysis of cardiac IL-6 expression (B). Semiquantitative analysis of trichrome staining in the heart (C) and the representative photos showing collagen deposition (indicated by arrows) around coronary arteries (D). Photos were taken at ×200. Scale bars=100 μm. *P<0.05 vs the PBS warm group; +P<0.01 vs the IL-6 shRNA cold group. Data=mean+SE. n=6.

Discussion

The present data demonstrated, for the first time, that chronic exposure to cold caused inflammation in kidneys, hearts, and blood vessels, as evidenced by increased levels of IL-6 and TNF-α expression and leukocyte infiltration. Interestingly, RNAi knockdown of IL-6 attenuated cold-induced inflammation and elevation of BP, suggesting that that inflammation plays a vital role in the pathogenesis of CIH and that IL-6 is a key mediator in this process. The finding is significant because of its potential to provide novel preventive and therapeutic strategies for cold-related cardiovascular and renal dysfunctions, which are important for people who live in cold regions and during the winter. The prolonged attenuation of CIH is likely to because of the long-term expression vector, AAV. AAV is an effective and nonpathogenic vector that has been used to deliver therapeutic genes to the cardiovascular system and kidneys for long-term control of hypertension.23,24,26 AAV can express for months after gene delivery.24,27

Our previous studies have established that overactivation of the renin-angiotensin system is responsible for the development of CIH. In addition to its vasoconstrictor properties, angiotensin II has been demonstrated to exert proinflammatory effects in the vasculature by inducing production of integrins, adhesion molecules, cytokines, and growth factors through activation of the transcription factor nuclear factor-κB.13,28 Nuclear factor-κB, in turn, regulates a variety of proinflammatory genes at the transcriptional level.29 Angiotensin II has also been shown to directly contribute to the upregulation of IL-6 and has the ability to influence various stages of the inflammatory process.14,30–33 It will be interesting to test whether inflammation mediates the role of the renin-angiotensin system in CIH.

The molecular mechanisms involved in the pathogenesis of hypertension are not fully understood. Inflammation has been shown to increase the generation of reactive oxygen species by activating NADPH oxidases.34 IL-6 is a strong activator of NADPH oxidases.35,36 Normal superoxide production via NADPH oxidases is necessary for biological processes, such as cell signaling, posttranslational modification, and host defense.34 An overproduction of reactive oxygen species, such as superoxide, however, can lead to oxidative damage in the vasculature resulting in endothelial dysfunction, vascular senescence and remodeling, and hypertension.34,37 The present study clearly showed that knockdown of IL-6 led to a decrease in cold-induced vascular superoxide production, which may contribute to its antihypertensive effect.

Patients with cardiovascular disease have increased protein expression and plasma concentration of many inflammatory markers, including selectins (P, E, and L selectins), intracellular adhesion molecule 1, and vascular cell adhesion molecule.38 Human hypertension is associated with increased levels of TNF-α and IL-6, as well as intracellular adhesion molecule 1, vascular cell adhesion molecule, and selectins.39 However, the role of IL-6 in inflammation in the context of hypertension is largely unknown. The present study revealed that IL-6 is likely a key mediator of inflammation, because knockdown of IL-6 prevented the cold-induced increases in TNF-α and leukocyte infiltration. The molecular mechanism leading to increased levels of macrophages and T cells in cold-exposed animals needs to be further investigated, but increased levels of IL-6 could upregulate intracellular adhesion molecule 1, vascular cell adhesion molecule, and the selectins through nuclear factor-κB activation leading to the recruitment of inflammatory infiltrates. These inflammatory factors are directly responsible for the recruitment of leukocytes, including macrophages and lymphocytes, to the vasculature and have been demonstrated to be upregulated in several animal models of hypertension.38,40–43

In the present study, systolic BP was monitored using the tail-cuff method. The tail-cuff procedure is a common method used by our laboratory12,23 and others44–45 to delineate CIH. It has been confirmed by the intra-arterial cannulation that the noninvasive tail-cuff method is effective and reliable in monitoring BP responses to cold.8,9,46

It is noted that chronic exposure to cold for 8 weeks is sufficient to cause glomerular collapse, a sign of renal structural damage. RNAi knockdown of IL-6 effectively prevented cold-induced glomerular damage, suggesting that IL-6 may mediate inflammatory responses that lead to renal vascular damage and, ultimately, glomerular collapse. Previous studies from our laboratory have demonstrated that chronic cold exposure also leads to cardiac hypertrophy.8,12,47 The mechanism of cold-induced cardiac hypertrophy is unknown but is independent of high BP, because prevention of CIH does not attenuate the development of cold-induced cardiac hypertrophy.1,12 The present data revealed that inflammation may play a role in the pathogenesis of cold-induced cardiac hypertrophy, because knockdown of IL-6 attenuated the cold-induced increase in heart weight. Interestingly, cold exposure also resulted in formation of fibrosis around the coronary artery, which may ultimately impair the coronary circulation. It is notable that cold-induced formation of fibrosis was abolished by inhibition of IL-6, suggesting an important role for inflammation in this pathological process.

TNF-α is a primary proinflammatory cytokine that has been shown to increase the production of cytokines, including IL-6, and to initiate inflammatory cascades.48,49 Both TNF-α and IL-6 blockade therapies can reduce inflammation and improve prognosis in patients with arthritis, a chronic inflammatory disease.50 Although TNF-α blockers have been shown to decrease IL-6 levels, this is the first study demonstrating that knockdown of IL-6 prevented cold-induced upregulation of TNF-α. Additional investigation into the molecular mechanism mediating the regulation of TNF-α by IL-6 would be of particular interest.

Perspectives

The current study yields 2 major findings in the pathogenesis of CIH. First, chronic exposure to cold caused inflammation in the cardiovascular system and kidneys, as evidenced by upregulation of proinflammatory cytokines (IL-6 and TNF-α) and an increase in leukocyte infiltration. Second, RNAi knockdown of IL-6 abolished cold-induced inflammation and attenuated cold-induced elevation of BP and organ damage. These results suggest that inflammation plays a critical role in the development of CIH and that IL-6 is a critical mediator in this pathological process. This study also reveals that AAV.RNAi knockdown of IL-6 may serve as a new approach for the effective control of hypertension.

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health grant R01 NHLBI-077490.

Disclosures

None.

  • Received November 1, 2009.
  • Revision received November 17, 2009.
  • Accepted March 18, 2010.

References

  1. ↵
    Sun Z. Cardiovascular responses to cold exposure. Front Biosci (Elite Ed). 2010; 2: 495–503.
    OpenUrlPubMed
  2. ↵
    Baker-Blocker A. Winter weather and cardiovascular mortality in Minneapolis-St. Paul. Am J Pub Health. 1982; 72: 261–265.
    OpenUrlCrossRefPubMed
  3. ↵
    Seretakis D, Lagiou P, Lipworth L, Signorello LB, Rothman KJ, Trichopoulos D. Changing seasonality of mortality from coronary heart disease. JAMA. 1997; 278: 1012–1014.
    OpenUrlCrossRefPubMed
  4. ↵
    Sheth T, Nair C, Muller J, Yusuf S. Increased winter mortality from acute myocardial infarction and stroke: the effect of age. J Am Coll Cardiol. 1999; 33: 1916–1919.
    OpenUrlCrossRefPubMed
  5. ↵
    Verdon F, Boudry JF, Chuat M, Studer JP, Truong CB, Jacot E. Seasonal variations in arterial pressure in hypertensive patients [in German]. Schweiz Med Wochenschr. 1993; 123: 2363–2369.
    OpenUrlPubMed
  6. ↵
    Minami J, Kawano Y, Ishimitsu T, Yoshimi H, Takishita S. Seasonal variations in office, home and 24 h ambulatory blood pressure in patients with essential hypertension. J Hypertens. 1996; 14: 1421–1425.
    OpenUrlCrossRefPubMed
  7. ↵
    Hata T, Ogihara T, Maruyama A, Mikami H, Nakamaru M, Naka T, Kumahara Y, Nugent CA. The seasonal variation of blood pressure in patients with essential hypertension. Clin Exp Hypertens. 1982; 4: 341–354.
    OpenUrl
  8. ↵
    Fregly MJ, Kikta DC, Threatte RM, Torres JL, Barney CC. Development of hypertension in rats during chronic exposure to cold. J Appl Physiol. 1989; 66: 741–749.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Sun Z, Cade R, Katovich MJ, Fregly MJ. Body fluid distribution in rats with cold-induced hypertension. Physiol Behav. 1999; 65: 879–884.
    OpenUrlCrossRefPubMed
  10. ↵
    Sun Z, Cade JR, Fregly MJ. Cold-induced hypertension: a model of mineralocorticoid-induced hypertension. Ann N Y Acad Sci. 1997; 813: 682–688.
    OpenUrlPubMed
  11. ↵
    Sun Z, Bello-Roufai M, Wang X. RNAi inhibition of mineralocorticoid receptors prevents the development of cold-induced hypertension. Am J Physiol Heart Circ Physiol. 2008; 294: H1880–H1887.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Sun Z, Cade R, Zhang Z, Alouidor J, Van H. Angiotensinogen gene knockout delays and attenuates cold-induced hypertension. Hypertension. 2003; 41: 322–327.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Marchesi C, Paradis P, Schiffrin EL. Role of the renin-angiotensin system in vascular inflammation. Trends Pharmacol Sci. 2008; 29: 367–374.
    OpenUrlCrossRefPubMed
  14. ↵
    Moriyama T, Fujibayashi M, Fujiwara Y, Kaneko T, Xia C, Imai E, Kamada T, Ando A, Ueda N. Angiotensin II stimulates interleukin-6 release from cultured mouse mesangial cells. J Am Soc Nephrol. 1995; 6: 95–101.
    OpenUrlAbstract
  15. ↵
    Androulakis ES, Tousoulis D, Papageorgiou N, Tsioufis C, Kallikazaros I, Stefanadis C. Essential hypertension: is there a role for inflammatory mechanisms?. Cardiol Rev. 2009; 17: 216–221.
    OpenUrlCrossRefPubMed
  16. ↵
    Di Bona D, Vasto S, Capurso C, Christiansen L, Deiana L, Franceschi C, Hurme M, Mocchegiani E, Rea M, Lio D, Candore G, Caruso C. Effect of interleukin-6 polymorphisms on human longevity: a systematic review and meta-analysis. Ageing Res Rev. 2009; 8: 36–42.
    OpenUrlCrossRefPubMed
  17. ↵
    Mima T, Nishimoto N. Clinical value of blocking IL-6 receptor. Curr Opin Rheumatol. 2009; 21: 224–230.
    OpenUrlCrossRefPubMed
  18. ↵
    Boos CJ, Lip GY. Is hypertension an inflammatory process? Curr Pharm Des. 2006; 12: 1623–1635.
    OpenUrlCrossRefPubMed
  19. ↵
    Vazquez-Oliva G, Fernandez-Real JM, Zamora A, Vilaseca M, Badimon L. Lowering of blood pressure leads to decreased circulating interleukin-6 in hypertensive subjects. J Hum Hypertens. 2005; 19: 457–462.
    OpenUrlCrossRefPubMed
  20. ↵
    Manabe S, Okura T, Watanabe S, Fukuoka T, Higaki J. Effects of angiotensin II receptor blockade with valsartan on pro-inflammatory cytokines in patients with essential hypertension. J Cardiovasc Pharmacol. 2005; 46: 735–739.
    OpenUrlCrossRefPubMed
  21. ↵
    Fliser D, Buchholz K, Haller H. Antiinflammatory effects of angiotensin II subtype 1 receptor blockade in hypertensive patients with microinflammation. Circulation. 2004; 110: 1103–1107.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Gadonski G, LaMarca BB, Sullivan E, Bennett W, Chandler D, Granger JP. Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of interleukin 6. Hypertension. 2006; 48: 711–716.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Wang X, Skelley L, Cade R, Sun Z. AAV delivery of mineralocorticoid receptor shRNA prevents progression of cold-induced hypertension and attenuates renal damage. Gene Ther. 2006; 13: 1097–1103.
    OpenUrlCrossRefPubMed
  24. ↵
    Wang Y, Sun Z. Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage. Hypertension. 2009; 54: 810–817.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Feng M, Whitesall S, Zhang Y, Beibel M, D'Alecy L, DiPetrillo K. Validation of volume-pressure recording tail-cuff blood pressure measurements. Am J Hypertens. 2008; 21: 1288–1291.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Phillips MI, Mohuczy-Dominiak D, Coffey M, Galli SM, Kimura B, Wu P, Zelles T. Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense. Hypertension. 1997; 29: 374–380.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Kimura B, Mohuczy D, Tang X, Phillips MI. Attenuation of hypertension and heart hypertrophy by adeno-associated virus delivering angiotensinogen antisense. Hypertension. 2001; 37: 376–380.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Touyz RM. Molecular and cellular mechanisms in vascular injury in hypertension: role of angiotensin II. Curr Opin Nephrol Hypertens. 2005; 14: 125–131.
    OpenUrlCrossRefPubMed
  29. ↵
    Savoia C, Schiffrin EL. Inflammation in hypertension. Curr Opin Nephrol Hypertens. 2006; 15: 152–158.
    OpenUrlPubMed
  30. ↵
    Funakoshi Y, Ichiki T, Takeda K, Tokuno T, Iino N, Takeshita A. Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells. J Biol Chem. 2002; 277: 18710–18717.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Kandalam U, Clark MA. Angiotensin II activates JAK2/STAT3 pathway and induces interleukin-6 production in cultured rat brainstem astrocytes. Regul Pept. 2010; 159: 110–116.
    OpenUrlCrossRefPubMed
  32. ↵
    Wu J, Yang X, Zhang YF, Zhou SF, Zhang R, Dong XQ, Fan JJ, Liu M, Yu XQ. Angiotensin II upregulates Toll-like receptor 4 and enhances lipopolysaccharide-induced CD40 expression in rat peritoneal mesothelial cells. Inflamm Res. 2009; 58: 473–482.
    OpenUrlCrossRefPubMed
  33. ↵
    Ruef J, Browatzki M, Pfeiffer CA, Schmidt J, Kranzhofer R. Angiotensin II promotes the inflammatory response to CD40 ligation via TRAF-2. Vasc Med. 2007; 12: 23–27.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Sedeek M, Hebert RL, Kennedy CR, Burns KD, Touyz RM. Molecular mechanisms of hypertension: role of Nox family NADPH oxidases. Curr Opin Nephrol Hypertens. 2009; 18: 122–127.
    OpenUrlCrossRefPubMed
  35. ↵
    Behrens MM, Ali SS, Dugan LL. Interleukin-6 mediates the increase in NADPH-oxidase in the ketamine model of schizophrenia. J Neurosci. 2008; 28: 13957–13966.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Volk T, Hensel M, Schuster H, Kox WJ. Secretion of MCP-1 and IL-6 by cytokine stimulated production of reactive oxygen species in endothelial cells. Mol Cell Biochem. 2000; 206: 105–112.
    OpenUrlCrossRefPubMed
  37. ↵
    Sedeek M, Gilbert JS, LaMarca BB, Sholook M, Chandler DL, Wang Y, Granger JP. Role of reactive oxygen species in hypertension produced by reduced uterine perfusion in pregnant rats. Am J Hypertens. 2008; 21: 1152–1156.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Savoia C, Schiffrin EL. Vascular inflammation in hypertension and diabetes: molecular mechanisms and therapeutic interventions. Clin Sci (Lond). 2007; 112: 375–384.
    OpenUrlCrossRefPubMed
  39. ↵
    Preston RA, Ledford M, Materson BJ, Baltodano NM, Memon A, Alonso A. Effects of severe, uncontrolled hypertension on endothelial activation: soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1 and von Willebrand factor. J Hypertens. 2002; 20: 871–877.
    OpenUrlCrossRefPubMed
  40. ↵
    Haverslag R, Pasterkamp G, Hoefer IE. Targeting adhesion molecules in cardiovascular disorders. Cardiovasc Hematol Disord Drug Targets. 2008; 8: 252–260.
    OpenUrlCrossRefPubMed
  41. ↵
    Muller WA. Mechanisms of transendothelial migration of leukocytes. Circ Res. 2009; 105: 223–230.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    De Ciuceis C, Amiri F, Brassard P, Endemann DH, Touyz RM, Schiffrin EL. Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-infused macrophage colony-stimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury. Arterioscler Thromb Vasc Biol. 2005; 25: 2106–2113.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Ando H, Zhou J, Macova M, Imboden H, Saavedra JM. Angiotensin II AT1 receptor blockade reverses pathological hypertrophy and inflammation in brain microvessels of spontaneously hypertensive rats. Stroke. 2004; 35: 1726–1731.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Peng JF, Kimura B, Fregly MJ, Phillips MI. Reduction of cold-induced hypertension by antisense oligodeoxynucleotides to angiotensinogen mRNA and AT1-receptor mRNA in brain and blood. Hypertension. 1998; 31: 1317–1323.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Zhu Z, Zhu S, Zhu J, van der Giet M, Tepel M. Endothelial dysfunction in cold-induced hypertensive rats. Am J Hypertens. 2002; 15: 176–180.
    OpenUrlCrossRefPubMed
  46. ↵
    Wang X, Sun Z. RNAi silencing of brain klotho potentiates cold-induced elevation of blood pressure via the endothelin pathway. Physiol Genomics. In press.
  47. ↵
    Sun Z, Fregly MJ, Cade JR. Effect of renal denervation on elevation of blood pressure in cold-exposed rats. Can J Physiol Pharmacol. 1995; 73: 72–78.
    OpenUrlPubMed
  48. ↵
    Desmet C, Warzee B, Gosset P, Melotte D, Rongvaux A, Gillet L, Fievez L, Seumois G, Vanderplasschen A, Staels B, Lekeux P, Bureau F. Pro-inflammatory properties for thiazolidinediones. Biochem Pharmacol. 2005; 69: 255–265.
    OpenUrlCrossRefPubMed
  49. ↵
    Navarro-Gonzalez JF, Mora-Fernandez C. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol. 2008; 19: 433–442.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest. 2008; 118: 3537–3545.
    OpenUrlCrossRefPubMed
View Abstract
Back to top
Previous ArticleNext Article

This Issue

Hypertension
June 2010, Volume 55, Issue 6
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • References
  • Figures & Tables
  • Supplemental Materials
  • Info & Metrics
  • eLetters

Article Tools

  • Print
  • Citation Tools
    Ribonucleic Acid Interference Knockdown of Interleukin 6 Attenuates Cold-Induced Hypertension
    Patrick Crosswhite and Zhongjie Sun
    Hypertension. 2010;55:1484-1491, originally published May 19, 2010
    https://doi.org/10.1161/HYPERTENSIONAHA.109.146902

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Hypertension.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    Ribonucleic Acid Interference Knockdown of Interleukin 6 Attenuates Cold-Induced Hypertension
    (Your Name) has sent you a message from Hypertension
    (Your Name) thought you would like to see the Hypertension web site.
  • Share on Social Media
    Ribonucleic Acid Interference Knockdown of Interleukin 6 Attenuates Cold-Induced Hypertension
    Patrick Crosswhite and Zhongjie Sun
    Hypertension. 2010;55:1484-1491, originally published May 19, 2010
    https://doi.org/10.1161/HYPERTENSIONAHA.109.146902
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects

  • Cardiology
    • Etiology
      • Hypertension
        • Hypertension
  • Heart Failure and Cardiac Disease
    • Remodeling
  • Basic, Translational, and Clinical Research
    • Animal Models of Human Disease

Hypertension

  • About Hypertension
  • Instructions for Authors
  • AHA CME
  • Guidelines and Statements
  • Permissions
  • Journal Policies
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Editorial Office Address:
7272 Greenville Ave.
Dallas, TX 75231
email: hypertension@heart.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured