A Team of Australian and German researchers has shown that miRNAs detected in conventional plasma preparations seem to be located primarily within microparticles, small vesicles released by several types of cells and linked to their parent cell type by distinct surface markers.
In a study published last week, the team also showed that microparticle-bound miRNAs can characterize different cardiovascular diseases by measuring distinctly different microparticle miRNA profiles between patients with acute and stable coronary artery disease.
The miRNAs in microparticles from several cell types also differed significantly from those in their parent cells, the team reported last week in Cardiovascular Research. Some miRNAs were upregulated in microparticles compared to maternal cells and others downregulated. This suggests that cells selectively package miRNAs into these vesicles, a potential mechanism for transferring gene regulatory function from one cell to another, the authors wrote.
The collaborators hail from the Baker IDI Heart and Diabetes Institute, Monash University, the University of Melbourne, and University Hospital Freiburg.
With research accelerating into blood-based miRNA signals as diagnostics for various cardiovascular diseases, the role of these cell-specific microparticles may be crucial to identifying specific and predictive miRNAs, the group suggested.
A central question is whether miRNAs detected in plasma studies may actually originate in these microparticles, the group wrote.
Because plasma contains microparticles, “we postulated that microparticles could be the source of miRNA in circulation," Peter Karlheinz and Philipp Diehl, two of the paper's authors, wrote in an e-mail to Gene Silencing News.
Having found a paucity of miRNA outside of microparticles and in light of evidence of selective transport of miRNAs from stimulated cells, the researchers argued their postulation seemed likely.
"We could demonstrate that microparticles contain a significantly distinct miRNA pattern compared to their corresponding stimulated and non-stimulated maternal cells, suggesting a selective miRNA transport into microparticles," the authors reported.
"Several of the miRNAs up-regulated in microparticles are involved in cardiovascular disease, indicating that microparticles may act as transport vehicles delivering specific miRNAs. Since microparticles are major constituents of typical plasma preparations, 'plasma' miRNA profiles attributed to specific disease may in fact originate from circulating microparticles," the researchers wrote.
According to Karlheinz and Diehl, the group was interested in establishing whether microparticles might be responsible for protecting miRNAs from enzymatic degradation in circulating blood because of rapidly advancing work using blood-based miRNA in the diagnosis of a number of cardiovascular diseases.
"As several cardiovascular diseases are associated with vascular inflammation, it has been shown that microparticles are increased in the blood of many of these diseases and furthermore contribute to their progression, However, most researchers accredit the pro-inflammatory effects of microparticles to their specific lipid composition," Karlheinz and Diehl wrote.
Meanwhile, "miRNA have been described in plasma of different cardiovascular [diseases] and other diseases. However, plasma contains high amounts of RNase and thus it remained an open question how miRNA can survive in plasma," the two noted in their e-mail.
The group used next-generation sequencing and qRT-PCR to compare miRNA profiles of microparticles with the profiles of their maternal cells in both a stimulated and non-stimulated state, and to compare the concentration of miRNA in plasma microparticles with the concentration of miRNA in particle-free plasma.
"The predominant amount of plasma miRNAs was found to be associated to microparticles and only [a] small amount were detected in microparticle-free plasma," the authors reported.
To investigate whether cells selectively pack their vesicles with miRNA, the group examined platelets and used THP-1 cells and human umbilical vein endothelial cells, or HUVECs, as models for monocytes and endothelial cells to avoid contamination by other cell types.
In platelets, the group found that several miRNAs were significantly increased in platelet microparticles over platelets themselves, while two — miRNA-126 and miRNA-133 — were higher in platelets than in their microparticles.
The group measured miRNAs of THP-1 cells in both stimulated and unstimulated states against their associated microparticles and found that miRNAs in THP-1 microparticles differed from those in both stimulated and unstimulated parent cells. Moreover, cells appeared to package a different group of miRNAs depending on their stimulation state, the researchers reported.
Among miRNAs increased in microparticles from stimulated cells, the group found several known pro-inflammatory miRNAs.
The researchers saw similar results in HUVECs, with stimulated endothelial cells showing different miRNA profiles and appearing to "pack their microparticles with distinct miRNAs," they wrote.
"We described that microparticles contain a different miRNA pattern than [their] maternal cells. Hence, we suggested that there needs to be a specific loading mechanism by which microparticles are packed with distinct miRNA," Dietz and Karlheinz wrote.
"However, it is currently unknown how such a loading or packaging mechanism could work,” they added. “Indeed, our work is the first really to postulate such a mechanism."
The researchers then measured microparticle miRNA in a small cohort of patients with acute and stable coronary artery disease. They found that five patients with stable CAD had significantly less microparticle-associated miRNAs than another five with acute CAD.
Karlheinz and Dietz said in their email that the group is planning to try next to define specific disease-related patterns of miRNA in microparticles.
The Melbourne team is not alone in this direction. A group of Chicago researchers searching for circulating miRNAs as potential biomarkers in breast cancer recently published a study outlining ways to optimize sample preparation for quantifying miRNAs in plasma, but said such methods would not even be necessary when profiling miRNAs from breast cancer-specific vesicles (GSN 12/22/11).
"By going clearly after nanovesicles that we know by surface markers specifically came from, in our case, breast cancer, we can become more specific," one of the authors, Dominik Duelli, told Gene Silencing News at the time. "You avoid the entire problem [of blood sample preparation] completely because if you have an antibody against your specific vesicle, you remove all this other stuff," he said.
Additionally, Dietz and Karlheinz plan to work on constructing artificial microparticles as "unique therapeutic vehicles," the two wrote in their email. "You can envisage using artificial microparticles to be used as vehicles for therapeutic miRNAs," they said.
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