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HIF-1α overexpression and experimental murine atherosclerosis

Interview with senior author Professor George, Tel Aviv Sourasky Medical Center
Basic Sciences, Pharmacology, Genomics and Cardiovascular Pathology


Q: Professor George, a very interesting article of your team is published in this month’s edition of ATVB. What are the key results of this study?

A: In the published paper we demonstrate that the hypoxia inducible gene HIF-1α can be used to modulate the immune system and by forcing its expression in lymphocytes, we were able to shift the immune system to a T helper-2 program (i.e an antiinflammatory pattern). This program was evident by a reduced expression of inflammatory cytokines such as IFN-γ and TNF-α, together with an induction of anti-inflammatory cytokines, such as IL-10. We show here that forcing HIF2 expression into lymphocytes was associated with inhibition of atherosclerotic plaque progression and intra-plaque IFN-γ expression, in ApoE knockout mice.


Q: Indeed, there are a number of elements in this study we can talk about more extensively. For instance, the plasmid you used encoded for a “special” form of HIF-1α, would you mind explaining this in greater detail?

A: In order to induce HIF-1α constitutive expression in splenocytes, we employed a plasmid encoding a mutant form of HIF-1α, HIF-1αP564A. As mentioned in the text, HIF-1α subunits degradation depends on the hydroxylation of specific proline sites by prolyl hydroxylases, to allow VHL protein binding and subsequent ubiquitination. The HIF-1αP564A mutant contains a point mutation consisting of proline to alanine in aa 564 in the pVHL binding motif, leading to enhanced HIF-1α stability.


Q: As the HIF-1α was taken out of the usual control sequence of hydroxylation, VHL protein binding, ubiquitination, and degradation via the proteasome complex, how do you envision “constitutive” activation of HIF-1α in the atherosclerotic plaque in humans? Is the oxygen milieu of the atherosclerotic plaque an important factor and/or increased oxidative stress? Could the effects of increased HIF-1α expression under those circumstances differ from the ones seen in the current study?

A: HIF-1α expression within the atherosclerotic plaque was previously described by several researchers. In these studies HIF-1α expression within the hypoxic plaque, especially in macrophage environment, was found to be correlated with accelerated plaque progression and neovascularization. Our study focused on systemic modification of lymphocyte programming by affecting the major splenic pool of these cells. This was achieved by repeated short-term overexpression of HIF-1α in CD4+ lymphocyte. Eventually, this systemic modulation resulted in reduced aortic expression of inflammatory cytokine expression and inhibited plaque progression.


Q: Does it need constitutive activity of HIF-1α to see the study effects or would intermittent “spurs” suffice?

A: In fact, the hydrodynamic DNA injection used in our study to drive HIF-1α overexpression induced short-term expression via in-vivo transient transfection. As mentioned, the injections were repeated twice before sacrifice. The induced effects on lymphocyte and cytokine expression maintained a significantly longer plasmid transcription. A very provocative and implementive question would be how whole body hypoxia would influence atherosclerosis progression. We can not say that our results support the fact that people living in areas with reduced oxygen tension are more likely to have less atherosclerosis since hypoxia controls a cluster of genes, not just hypoxia inducible factor. Yet, this is an exciting question that can be partially answered by large epidemiologic studies.


Q: The pathomechanistic consequences of HIF-1α on attenuating atherosclerosis seem to be linked to the leukocytes, particularly a shift in the Th1 to Th2 population of the atherosclerotic plaque. Would you mind explaining in more detail how HIF-1α regulates this shift?

A: In addition to its role as regulator of the cellular response to hypoxic conditions, multiple regulatory functions were linked to HIF-1α. Regarding HIF-1α immunomodulatory roles, it was previously found that HIF-1α controls the production of inflammatory cytokines such as; IFN- , TNF- , IL-2, IL-4, and IL-13. It was also demonstrated that a negative regulation of HIF-1α exists on lymphocyte NF-КB-mediated program. My lab has recently demonstrated a positive correlation between HIF-1α expression and occurrence/activity of Foxp3+CD4+CD25+ T-regulatory cells. The exact mechanisms by which these regulatory programs evolve still remain to be further elucidated. In our study, we have shown attenuation of lymphocyte TH1 characteristic cytokines in vivo, accompanied by an elevation of TH2 related cytokine expression. The molecular basis of these effects could be via cross talk of HIF with transcription factors that control T-helper programs like the STATs. We have also very recently shown that HIF can drive an anti inflammatory program in lymphocytes by upregulating the master transcriptional regulator of regulatoryT cells namely FoxP3.


Q: One may wonder about HIF-1α-induced increased in VEGF expression and neovessel formation in the atherosclerotic plaque. Would this be a possible mechanism by which HIF-1α could actually contribute to atherosclerotic plaque development, i.e. the opposite effect to the one described in the current study?

A: As mentioned above, intra-plaque expression of HIF-1α was indeed documented and apparently occurs as a result of local hypoxia. Vink et al (2007) found ensuing effects on VEGF expression and neovascularization. However, our study focused on the effect of HIF-1α-mediated modification of lymphocytes on plaque progression. Our RT-PCR analysis examines cytokine expression in splenocytes and aortas at different time point, mainly remote from plasmid injection. We found that attenuation of inflammatory cytokine production, achieved by lymphocyte expression of HIF-1α, reduces lipid core enlargement.

With regard to plaque vascularization, the data in the literature is mixed with regard to its potential effects on plaque progression and stability. Several reports support a proatherogenic effect for increased vascularity whereas others show that it may act the other way. I believe that until we obtain a more firm idea of what VEGF and consequent angio/vasculogenesis does to the plaque we cannot use these as surrogates to prove mechanistic insights of plaque growth


Q: With neovessel formation, could plaque hypoxia be decreased to such an extent that HIF-1α expression decreases and what would be the ultimate consequence – pro- or anti-atherosclerotic?

A: This is a complex issue; local angiogenesis may actually increase the local oxygen levels, affecting HIF-1α expression. On the other hand, micro-vessel formation strongly contributes to the homing of immune cells and thus local inflammation. Additionally, the accumulating data about HIF-1α mechanism of expression and transcription reveal its involvement in multiple complex processes including immunomodulation and stress response.


Q: Could these considerations have clinical implications, and if so, how should we target HIF-1α? Previous studies indicated the potential of statin drugs in this area. There is also some mentioning about aspirin’s potential to inhibit the proteasome, which would lead to increased HIF-1α expression.

A: These approaches are indeed potentially promoting HIF-1α induction. However, a panel of additional associated and non-associated genes is driven by these small molecules and it would be probably impossible to dissect the potential role of HIF. Nevertheless I do regard this as a potential mediator of the effect of the drugs you mentioned and perhaps derivatives of statins or asprin with improved effects on HIF could be more potent in vivo. This is indeed a very provocative and implementive prospect.


Q: What are the necessary next studies in this area? How should the HIF-1α story continue?

A: Recently, HIF-1α regulatory roles turn out to be complex and multidirectional. Thus, the effects of HIF-1α activity on lymphocyte stimulation should be further studied. One of our next steps might be selective modulations of HIF-1α in specific populations of T cells to achieve efficient cell transfer to treat atherosclerosis and to allow an accurate assessment of the mechanisms and functions that affect plaque progression. It would also be interesting to gain further insight into the downstream programs driven by HIF in lymphocytes and possibly study the role this master transcriptional factor in antigen presenting cells with regard to atherosclerosis.




Conclusion:

Q: This was an extraordinary experience and privilege to discuss this topic with you. If anyone has additional questions, suggestions, or comments, could they direct it to my E-mail address herrmann.joerg@mayo.edu or yours?

Thank you so much, Professor George.

A: It was my pleasure. I will be happy to answer to questions and receive comments and suggestions regarding our study jacobg@post.tau.ac.il

Thank you very much for your interest.

References


Arterioscler Thromb Vasc Biol. 2009 May; 29(5):665-70
The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.

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