Donald J. McCrann

A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and in-house platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by quantitative real-time (QRT)-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent preprocessing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.

The A2b adenosine receptor protects against vascular injury

The A2b adenosine receptor (A2bAR) is highly abundant in bone marrow macrophages and vascular smooth muscle cells (VSMC). To examine the functional significance of this receptor expression, we applied a femoral artery injury model to A2bAR knockout (KO) mice and showed that the A2bAR prevents vascular lesion formation in an injury model that resembles human restenosis after angioplasty. While considering related mechanisms, we noted higher levels of TNF-α, an up-regulator of CXCR4, and of VSMC proliferation in the injured KO mice. In accordance, CXCR4, which is known to attract progenitor cells during tissue regeneration, is up-regulated in lesions of the KO mice. In addition, aortic smooth muscle cells derived from A2bAR KO mice display greater proliferation in comparison with controls. Bone marrow transplantation experiments indicated that the majority of the signal for lesion formation in the null mice originates from bone marrow cells. Thus, this study highlights the significance of the A2bAR in regulating CXCR4 expression in vivo and in protecting against vascular lesion formation.

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence.

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16INK4a and senescence‐associated β‐galactosidase activity are detected primarily in the old tissues. VSMC from p16INK4a knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.

Differential expression of NADPH oxidases in megakaryocytes and their role in polyploidy

Megakaryocytes (MKs) undergo an endomitotic cell cycle, leading to polyploidy. We examined the expression of the flavoproteins and oxidative stress-promoting enzymes, NADPH oxidases (Noxs), in MKs because of their known role in promoting the cell cycle. Although the expression of Nox isoforms varies between cell types, they are induced at the mRNA level by mitogenic stimuli. Western blotting or reverse transcription-polymerase chain reaction of purified mouse MKs isolated from thrombopoietin (TPO)-treated bone marrow (BM) cultures indicated high expression of Nox1, a weak expression of Nox4, and no significant expression of Nox2. Immunofluorescence of freshly isolated MKs confirmed strong expression of Nox1 in one-third of MKs, whereas Nox1 staining was detected in nearly all MKs in TPO-stimulated BM cultures. Treatment of mouse BM cultures with Nox inhibitors resulted in accumulation of MKs with low DNA content levels and significant reduction of higher ploidy MKs. Purified, Nox-inhibited MKs showed a notable decrease in the level of the G(1) phase cyclin E, a cyclin associated with MK polyploidy, and its up-regulation restored most of the effect of Nox inhibitors. Hence, this study shows the expression of Nox isoforms in MKs and highlights a potential role of flavoproteins in promoting polyploidization in this lineage.

Direct visualization of the endomitotic cell cycle in living megakaryocytes: Differential patterns in low and high ploidy cells

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (≥ 8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Vascular smooth muscle cell polyploidy: An adaptive or maladaptive response?

Polyploidy is a state in which a cell contains multiple copies of its entire genome, while a normal diploid cell contains only two sets of homologous chromosomes. Although widely studied and pervasive in nature, the signals and mechanisms of polyploidization and its accompanying operational consequences are still unclear. This review focuses on relevant questions in deciphering the regulation of polyploidization of vascular smooth muscle cells (VSMC) in mammals and the role of polyploidy in various vascular pathologies, such as hypertension and aging. Additionally, we will explore new investigations in polyploidization of VSMCs involving the rapidly expanding fields of oxidative stress and senescence. J. Cell. Physiol. 215: 588–592, 2008.

Survivin overexpression alone does not alter megakaryocyte ploidy nor interfere with erythroid/megakaryocytic lineage development in transgenic mice

The level of survivin was reported to be scarce in mouse megakaryocytes (MKs) compared with erythroid cells. Considering this finding and previously reported in vitro data showing decreased MK ploidy upon retroviral-mediated overexpression of survivin, we sought to examine whether ectopic survivin expression in the MK lineage might alter ploidy level in vivo. Here we report the generation of 2 tissue specific hematopoietic transgenic mouse models, one expressing survivin in both the erythroid and MK lineages and the other expressing survivin solely in the MK lineage. Survivin protein overexpression was confirmed in MKs and erythrocytes. Surprisingly, analysis of both transgenic mouse lines showed no detectable changes in MK number, ploidy level, and platelet and erythrocyte counts, as compared with control mice. We conclude that elevated survivin expression does not alter MK/erythroid lineage development and that elevated survivin, alone, does not interfere with MK ploidy in vivo.

Survivin localization during endomitosis of high ploidy mouse megakaryocytes.

To the editor:nnDuring polyploidization the megakaryocyte (MK) undergoes endomitosis, which involves skipping late anaphase and cytokinesis.[1][1],[2][2] As a regulator of anaphase and cytokinesis, our laboratory implicated survivin in this process due to the absence of survivin protein in