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Project's title: "MicroRNA signature of vascular cells cross-talk relevant for the atherosclerotic plaque rupture in patients with type II diabetes"


Project's code: PN-II-RU-TE-2014-4-0965

Contract no.: TE 34/1.10.2015

Funding source: State budget

Contract authority: Executive Agency or Higher Education Research Development and Innovation Funding (UEFISCDI)

Budget: 550.000 lei

Period: 1.10.2015-30.09.2017

Project’s manager Elena Butoi, Ph.D.

Project’s team:

Tucureanu (Pirvulescu) Monica-Madalina

Constantinescu Cristina Ana

Calin Manuela

Stan Daniela

Macarie Razvan Daniel

Niculescu Loredan Stefan

Mesca Gabriela



Patients with diabetes mellitus have a higher risk of cardiovascular (CVD) morbidity and mortality due to several-fold increased risk for atherosclerotic plaque rupture. Atheroma development implicate vascular resident and recruited cells and their secretory products that ultimately leads to plaque rupture and atherothrombosis. Vascular smooth muscle cells (SMCs) are responsible for extracellular matrix (ECM) secretion and fibrous cap formation during plaque development. Communication of SMCs with monocytes/ macrophages plays important roles in atherosclerosis evolution. Previously we showed that monocytes-SMC cross-talk induces an inflammatory phenotype of both cell types. In this project our goal is to search the effect of diabetic macrophage-SMC communication on ECM proteins and to identify the specific miRNA involved. These miRNAs will be next quantified in serum from patients with acute coronary syndrome (ACS) with/without diabetes. The objectives are: 1. To investigate the complex interplay among macrophage-SMC interaction and diabetic stimuli on modulation and function of ECM proteins; 2. Identification of specific miRNA modified by diabetic macrophage-SMC cross-talk that modulate the ECM proteins; 3. Detection of relevant miRNAs to patients with acute ACS with or without diabetes. Understanding the mechanisms involved in ECM modulation in diabetic conditions and the identification of specific miRNAs involved may provide novel therapeutic targets for CVD diseases.


Expected results:

Patients with diabetes mellitus have a considerably higher risk of CVD morbidity and mortality, having several-fold increased risk of atherosclerotic plaque rupture that lead to CVD events such as myocardial infarction and stroke. Finding of biomarkers related to atherosclerotic plaque vulnerability, the factor that dictates the plaque rupture and further thrombosis, will help to improve the prediction of risk for this disease.  A better understanding of the processes driving vascular remodeling in diabetes should help develop new therapies. Therefore, we will identify: (i) the effect of macrophage-SMC communication on matrix proteins expression and destruction; (ii) selective microRNAs regulated by vascular cells communication and will analyze their involvement in ECM proteins regulation.



Phase I

Period: 1.10.2015-30.12.2015

Objective 1. To investigate the complex interplay among macrophage-SMC interaction and diabetic stimuli on modulation and function of extracellular matrix (ECM) protein.

Task 1.1 Investigation of the effect of diabetic macrophage-SMC interaction on matrix proteins (collagen I, III and elastin) in both cell types

Summary of results obtained

In this phase, the co-culture system between macrophages and smooth muscle cells was realized, and cells were maintained in co-culture for 24h or 72h in normal glucose conditions (5.5mM) or high glucose conditions (25mM). The matrix proteins were investigated at the gene and protein expression by qRT-PCR and Western Blot. The showed that the co-culture between macrophages and SMC significantly decrease the gene expression of collagen I, III and elastin in SMC. When the co-culture were realised in high glucose conditions, for 72h, the collagen I and III was furthemore downregulated. The protein experession of collagen III was also significantly reduced in high glucose conditions, as reveald by Western Blot experiments. These results underline the important role of cell cross-talk on extracellular matrix proteins, and in the context of atherosclerotic plaque, the decreasion of these proteins lead to thining of atherosclerotic fibrous cap and plaque vulnerability. Moreover, the cell cross-talk in high glucose conditions apear to exacerbate this process of matrix destruction, beeng perhaps one of the reasons that in diabetes, atherosclerosis process is accelerated and more risky.


Phase II

Period: 30.12.2015 – 15.12.2016

Objective 1. To investigate the complex interplay among macrophage-SMC interaction and diabetic stimuli on modulation and function of extracellular matrix (ECM) protein.

Task 1.2 To search the ability of SMCs in normal versus diabetic conditions to construct a collagen fibril matrix from soluble precursors;

Task 1.3 to analyse the effect of diabetic macrophage-SMCs interaction on the collagenases expression (i.e. MMP1 and MMP13) and on activity of metalloprotease (i.e. MMP2 and MMP9);

Task 1.4 Examination of angiogenic capacity of the factors released by diabetic macrophage-SMCs co-culture;

Objective 2. Identification of specific miRNAs modified by diabetic macrophage-SMC cross-talk, which modulate the ECM proteins expression

Task 2.1 MiRNAs profile screening in conditioned media of diabetic macrophage-SMCs co-culture.

Summary of results obtained in phase II

In this phase, we investigated the effect of macrophages and smooth muscle cells cross-talk in normal or high glucose conditions on metalloproteins (collagenases and gelatinases) gene expression and enzymatic activity. Moreover, the angiogenic capacity of conditioned media and expression profiles of the miRNAs released by cells in condition media was also determined. The results showed that the co-culture between macrophages and SMC significantly increase the gene expression of MMP-1 collagenase and MMP-9 gelatinase in SMC. When the co-culture was realized in high glucose conditions, the expression of these metalloproteases was further induced. The enzymatic activity of gelatinase MMP-9 was also increased (13 times) in the conditioned media from SMC after their co-culture with diabetic macrophages. The number of new tube like structures is also increased by cell cross-talk, but not in high glucose conditions. The miRNA expression patterns in the conditioned media from control or interacted SMC and macrophages (as detected using miRNA microarray analysis by miRCURY LNA Array), found a different profile of microRNAs of control cells versus interacted cells. Using specific analysis (Venn logic diagrams), when all the commune microRNAs between samples were excluded, we found three specific microRNA released only by macrophages co-cultured in high glucose condition and 5 specific microRNA released only by SMC co-cultured in high glucose condition. These microRNAs will be further investigated for their role in MMPs expression and activity.

Since the collagenases and gelatinases gene and enzymatic activity are increased, and their activity is to destroy the collagens and other extracellular matrix elements, we may suppose that the cell cross-talk in high glucose conditions contribute to atherosclerotic plaque vulnerability.



Butoi E, Gan AM, Tucureanu MM, Stan D, Macarie RD, Constantinescu C, Calin M, Simionescu M, Manduteanu I, Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis, Biochim Biophys Acta. 2016 Apr 7; 1863(7 Pt A):1568-1578. (I.F. 5.128, AIS: 2.263)- articol premiat UEFISCDI

Book chapters:

Calin M, Butoi E, Manea SA, Simionescu M, Manea A (2016). Lessons from experimental-induced atherosclerosis – valuable for the precision medicine of tomorrow. In book: Arterial Revascularization of the Head and Neck: Text Atlas for Prevention and Management of Stroke, pp.341-365, Editor Horia Muresian, Springer, New York, DOI: 10.1007/978-3-319-34193-4_17, ISBN-10: 331934191X, ISBN-13: 978-3319341910 (Total Number 10 according Karlsruhe Virtual Catalogue)


1.Elena Butoi, Ana Maria Gan, Monica Madalina Pirvulescu, Daniela Stan, Cristina Ana Constantinescu, Manuela Calin, Maya Simionescu, Ileana Manduteanu. Soluble factors released by cross talk of macrophages with smooth muscle cells promotes extracellular matrix alterations and angiogenesis. 4th International Symposium on ADIPOBIOLOGY and ADIPOPHARMACOLOGY, October 28-31, 2015, Bucharest, communication awarded with “Best Poster”

2.Elena Butoi, Ileana Manduteanu, Maya Simionescu. Cross-talk between vascular and immune cells accelerates the evolution of atherosclerotic plaque: the mechanisms involved. The 11 ACADEMICIAN NICOLAE CAJAL SYMPOSIUM of THE ROMANIAN ACADEMY OF MEDICAL SCIENCES “Molecular Mechanisms of acute and chronic diseases””, Bucharest, march 17-19, 2016 – oral presentation

3.Razvan Daniel Macarie, Monica M. Tucureanua, Daniela Stan, Cristina Constantinescu, Manuela Calin, Ileana Manduteanu, Elena Butoi. Cross-talk between macrophages and smooth muscle cells in diabetic conditions impairs collagen and metalloprotease synthesis and activity. The 8-th national congress with international participants and 34-th annual scientific session of Romanian Society of Cellular Biology, June 8-12, 2016, Oradea, poster