Although the number of scientific publications in the field of CVD has grown exponentially over the last 10 - 12 years and so did our understanding of molecular mechanisms underlying atherosclerotic disease initiation and progression, advances in diagnostic and therapeutic opportunities did not match that pace. Surprisingly, skyrocketed investments into R&D made by pharmaceutical companies did not translate into the number of successfully launched new drugs, which largely remained static369-372.
In an attempt to identify the underlying causes of such an illogical lack of relationship between the increased number of candidates entering clinical trials and the flattened success rates reflected in novel drugs brought to the market, Astra Zeneca performed a longitudinal review of their 142 drug discovery projects active between 2005 – 2010 and ranging from preclinical studies to those that completed Phase II of clinical testing373. While in the preclinical and Phase I stages safety issues were the primary reason responsible for the 82%
and 62% project closures, respectively, lack of efficacy emerged as the key factor contributing to an average of approximately 72% of project failures in Phase II clinical trials.
Interviewing teams supervising the studies resulted in an interesting conclusion that despite the drug candidates transitioning to the next phases, in many cases the understanding of the target role in the disease pathology was very poor and sufficiently robust data was missing, being a likely effect of commercial value-driven approach of the company instead of one promoting strong scientific rationale. In combination with the observation that the genetic link of the target to the human disease is a strong predictor of successful projects, this highlights the necessity of solid foundations in combining 1) understanding of molecular biology behind the candidate target role; with 2) securing the relevance of such a target for the human setting. Integration of the two has been shown to be the single most crucial action the scientific team can take to assure the highest success rates of potential drug candidates.
While around 150-192 million animals are being used for research worldwide each year374, independent reviews show that most animal models do not reliably replicate human disease375, which results in up to 90% of all studies failing to progress into late-stage clinical trials 376. One of the possible explanations for such a phenomenon is that, despite DNA resemblance, genes identified to be of importance for the animal disease showed very few associations with the human equivalent and the role of many molecules could be only scarcely confirmed in humans375. Mice and rats are the most commonly used species in the modeling of CVD, however severe limitations are associated with these models. First and foremost, no spontaneous atherosclerosis development can be observed in rodents, therefore advanced plaques in most cases can only develop as a result of previous genetic modification.
It is an accelerated process, where within several weeks we observe late-stage lesions with profound differences when compared to their human counterparts, reflected in the lack of thick fibrous cap, only scarcely detected spontaneous plaque rupture or intraplaque hemorrhage and/or no calcification sheets, to mention a few377, 378. Rarely observed atherosclerosis-related manifestations of ischemia in mice are likely to a great extent explained by the fundamental discrepancies in the biology and vasculature morphology, including 1) significantly lower cholesterol levels; 2) different lipoprotein profile with a central role reserved for HDL; 3) healthy intima devoid of SMCs and composed only of ECs and internal elastic lamina; 4) less thick underlying media; 5) no vasa vasorum; and last, but not least 6) different predilection sites with aortic sinus and innominate artery being the preferential sites for atherosclerosis development; as compared to humans379-382. Altogether, although animal models provide an attractive in vivo setting for the mechanistic studies of potential therapeutic targets, it is important to acknowledge that they do not constitute faithful human disease representation and as such, in order to provide results with true translational value, require integration with the human disease.
With this perspective, of crucial notion is that all of the studies included in this PhD thesis originate from human disease, where the initial hypothesis generation was performed using a unique repository of late-stage human atherosclerotic lesions and patients’ data called the Biobank of Karolinska Endarterectomies (BiKE). Briefly, in silico analyses of clinical and molecular information allowed for the identification of novel candidate targets, which were further experimentally investigated and validated in order to gain a comprehensive understanding of their role in the disease (Figure 8). Such an integrative pipeline originating from the human biobank brings a possibility of extrapolation of the results to the human setting and assures the studies are performed with the perspective of future therapeutic potential.
Figure 8. BiKE workflow with identification of novel candidate targets directly from human carotid plaques, further extended into experimental characterization and validation of relevant molecules.
2 RESEARCH AIMS
The overarching objective of this thesis was to comprehensively investigate the molecular mechanisms involved in the control of SMC phenotype and function in vessel wall biology, pathology and in healing reactions. Special focus was devoted to SMC phenotypic modulation and transcriptional regulation of the different subphenotypes.
The specific research aims within particular projects were:
Study I. To identify novel genes specific for vascular SMCs and to understand the mechanisms involved in SMC phenotypic switch in atherosclerosis.
Study II. To elucidate the role of proprotein convertase subtilisin/kexin 6 (Pcsk6) in the control of SMC function in vascular remodeling.
Study III. To investigate the SMC-specific molecular mechanisms which underlie the atherosclerotic lesion characteristics measured with ultrasound.
Study IV. To functionally characterize the major transcriptional regulators in vulnerable plaques and their impact on SMC phenotypic modulation.
3 MATERIALS AND METHODS
Connecting patient clinical phenotype with an improved understanding of molecular mechanisms driving the disease may in the future yield novel diagnostic and therapeutic approaches with unprecedented impact on cardiovascular morbidity and mortality. To assure the translational potential of our studies, discovery and initial characterization of the research findings were performed using several complementary human biobanks. This was further extended into studies in human atherosclerotic lesions in situ, in vivo models of intimal hyperplasia formation and in vitro mechanistic investigations in human carotid SMCs and primary rat and mouse aortic SMCs (Figure 9).
Figure 9. Schematic representation of methodology used for studying SMC phenotypic modulation.
Hypotheses generation and initial in silico experiments were performed using multiple human biobanks encompassing normal control arteries, end-stage atherosclerotic lesions, as well as atheroprogression tissues ranging from stage I adaptive intimal thickening to stage V fibroatheromas. This was further complemented by functional and mechanistic in situ, in vivo, ex vivo,and in vitro studies aiming at unraveling complexity of SMC phenotypic modulation.