EC-specific Gene Expression Research Articles

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture.

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

LncRNA MALAT1 facilitates BM-MSCs differentiation into endothelial cells via targeting miR-206/VEGFA axis

ABSTRACT Bone marrow-derived mesenchymal stem cells (BM-MSCs) implantation shows a repair effect on erectile function in diabetes mellitus-induced erectile dysfunction (DMED) due to its differentiative capacity into endothelial cells (ECs) that contributes to endothelial repair. This study was designed to explore the functional role and mechanism of long noncoding RNA (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in BM-MSCs-mediated DMED repairing. The DMED rat model was established and the erectile function was evaluated by calculating the intracavernous pressure (ICP)/mean arterial pressure (MAP) ratio in the DMED models with or without BM-MSCs implantation. The differentiation of BM-MSCs toward ECs was assessed by measuring the expression of EC-specific genes. RNA pull-down and luciferase reporter assay were performed to explore the interaction between miR-206 and MALAT1 or VEGFA. BM-MSCs implantation improved the erectile function of DMED rats and increased MALAT1 expression. MALAT1 was time-dependently upregulated during the VEGF-induced BM-MSCs differentiation into ECs. Mechanistically, MALAT1 acted as a sponge of miR-206 to upregulate VEGFA expression, thereby promoting the differentiation of BM-MSCs into ECs. Moreover, MALAT1 silencing in vivo impaired the repairing effect of BM-MSCs on erectile dysfunction. Collectively, MALAT1 facilitates BM-MSCs differentiation into ECs via regulating miR-206/VEGFA axis.

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

Abstract 142: New Approach for Directly Reprogrammed Endothelial Cells

Rationale: For therapeutic neovascularization through cell therapy to treat cardiovascular diseases, recently, we successfully generated reprogrammed ECs, directly from human dermal fibroblasts (HDF) with the ETV2 transcription factor. Although functional ECs were generated, ETV2 was delivered lentiviral vector, which is clinically unsuitable. Objective: In this study, we aimed to generate clinically compatible rECs, by employing an adenoviral vector to minimize genetic integration. Furthermore, to prolong the cell retention and improve cell survival, we applied synthetic biomaterial, polypeptide amphiphile (PA) nanomatrix gel and tested for experimental cell therapy in animal models. Methods and Results: After we transduced HDFs with Ad- ETV2 (Ad- ETV2 -HDF), EC gene expression was measured by qRT-PCR and flow cytometry. We found that Ad- ETV2- HDFs displayed a cobblestone-like morphology and showed notable induction of EC genes (CDH5, KDR, PECAM1). Flow cytometry analysis showed peak expression of CDH5 and KDR at D6 and continuous increase of PECAM1 over 9 days. Also, Ad- ETV2 -HDFs took up acetylated LDL and bound lectin, suggesting that these cells possess functional EC characteristics. To enrich cells showing EC characters, Ad- ETV 2-HDFs were sorted for KDR by FACS at day 6. qRT-PCR showed substantial enrichment of EC-specific gene expression in KDR + cells. Only KDR + cells (reprogrammed ECs, rECs) formed tube-like structures on Matrigel. In addition, immunostaining confirmed high expression of EC marker proteins in rECs. Next, we tested the functionality of rECs in hindlimb ischemia mouse model. At 4 weeks after injection of 4 x 10 5 rECs into the ischemic hindlimb, we observed rECs incorporated into host blood vessels or resided perivascular regions. Furthermore, we found more rECs in the tissues and were directly incorporated into the vessels when they were encapsulated in PA nanomatrix gel compared to bare rEC-injected mice. Conclusions: Together, these data strongly support the generation of Ad- ETV2 -induced rECs (Ad-rECs) and enhanced retention of rECs by PA nanomatrix gel in tissue. With this study, we can develop advanced concept and techniques for clinical application of rECs and establish a new platform for future cell therapy.

Vascularization of Natural and Synthetic Bone Scaffolds

Vascularization of engineered bone tissue is critical for ensuring its survival after implantation. In vitro pre-vascularization of bone grafts with endothelial cells is a promising strategy to improve implant survival. In this study, we pre-cultured human smooth muscle cells (hSMCs) on bone scaffolds for 3 weeks followed by seeding of human umbilical vein endothelial cells (HUVECs), which produced a desirable environment for microvasculature formation. The sequential cell-seeding protocol was successfully applied to both natural (decellularized native bone, or DB) and synthetic (3D-printed Hyperelastic “Bone” scaffolds, or HB) scaffolds, demonstrating a comprehensive platform for developing natural and synthetic-based in vitro vascularized bone grafts. Using this sequential cell-seeding process, the HUVECs formed lumen structures throughout the DB scaffolds as well as vascular tissue bridging 3D-printed fibers within the HB. The pre-cultured hSMCs were essential for endothelial cell (EC) lumen formation within DB scaffolds, as well as for upregulating EC-specific gene expression of HUVECs grown on HB scaffolds. We further applied this co-culture protocol to DB scaffolds using a perfusion bioreactor, to overcome the limitations of diffusive mass transport into the interiors of the scaffolds. Compared with static culture, panoramic histological sections of DB scaffolds cultured in bioreactors showed improved cellular density, as well as a nominal increase in the number of lumen structures formed by ECs in the interior regions of the scaffolds. In conclusion, we have demonstrated that the sequential seeding of hSMCs and HUVECs can serve to generate early microvascular networks that could further support the in vitro tissue engineering of naturally or synthetically derived bone grafts and in both random (DB) and ordered (HB) pore networks. Combined with the preliminary bioreactor study, this process also shows potential to generate clinically sized, vascularized bone scaffolds for tissue and regenerative engineering.

ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation

Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation.

Endovascular Biopsy: In Vivo Cerebral Aneurysm Endothelial Cell Sampling and Gene Expression Analysis.

There is limited data describing endothelial cell (EC) gene expression between aneurysms and arteries partly because of risks associated with surgical tissue collection. Endovascular biopsy (EB) is a lower risk alternative to conventional surgical methods, though no such efforts have been attempted for aneurysms. We sought (1) to establish the feasibility of EB to isolate viable ECs by fluorescence-activated cell sorting (FACS), (2) to characterize the differences in gene expression by anatomic location and rupture status using single-cell qPCR, and (3) to demonstrate the utility of unsupervised clustering algorithms to identify cell subpopulations. EB was performed in 10 patients (5 ruptured, 5 non-ruptured). FACS was used to isolate the ECs and single-cell qPCR was used to quantify the expression of 48 genes. Linear mixed models and exploratory multilevel component analysis (MCA) and self-organizing maps (SOMs) were performed to identify possible subpopulations of cells. ECs were collected from all aneurysms and there were no adverse events. A total of 437 ECs was collected, 94 (22%) of which were aneurysmal cells and 319 (73%) demonstrated EC-specific gene expression. Ruptured aneurysm cells, relative controls, yielded a median p value of 0.40 with five genes (10%) with p values <0.05. The five genes (TIE1, ENG, VEGFA, MMP2, and VWF) demonstrated uniformly reduced expression relative the remaining ECs. MCA and SOM analyses identified a population of outlying cells characterized by cell marker gene expression profiles different from endothelial cells. After removal of these cells, no cell clustering based on genetic co-expressivity was found to differentiate aneurysm cells from control cells. Endovascular sampling is a reliable method for cell collection for brain aneurysm gene analysis and may serve as a technique to further vascular molecular research. There is utility in combining mixed and clustering methods, despite no specific subpopulation identified in this trial.

Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells.

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31+ population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.

Regulation of endothelial cell-specific Robo4 gene expression by DNA methylation and non-lineage specific transcription factors

Studies of tissue-specific gene expression have suggested that tissue-specific transcription factors or tissue-specific combinations of non-cell-type-specific transcription factors regulate tissue-specific gene expression. Although the studies of endothelial cell (EC)-specific gene expression has identified several transcriptional activators such as SP1, ETS family proteins, and GATA proteins, their expression and combinations are not likely to be EC-specific. To investigate the mechanism of EC-specific gene expression, we analyzed the regulation mechanism of an EC-specific gene, Roundabout4 (Robo4). We identified the 3-kb Robo4 promoter and several transcription factors including SP1 and GABP that bind to the Robo4 promoter and promote Robo4 gene expression. However, we could not explain the mechanism for EC-specific Robo4 gene expression with only those factors because their expression and combination are not EC-specific. Therefore, we hypothesized the contribution of other mechanisms, especially epigenetic control, to Robo4 gene regulation and demonstrated the importance of DNA methylation for EC-specific Robo4 expression. In this review, we summarize our recent studies and discuss the novel regulation model of Robo4 gene expression by transcription factors and DNA methylation.

The W-Loop of Alpha-Cardiac Actin Is Critical for Heart Function and Endocardial Cushion Morphogenesis in Zebrafish

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.

Abstract 42: In Vivo Differentiation of Epigenetically Reprogrammed Human Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Postinfarct Myocardial Repair

Currently, bone marrow derived endothelial progenitor cells (human CD34+ cells, EPC) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that CD34+ cells predominantly work through a paracrine mechanism, there exists no convincing evidence that these cells trans-differentiate into functional cardiomyocytes (CMC). Since ischemic heart disease leads to substantial loss of CMC, improving cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPC and CMC both differentiate from a common mesodermal progenitor however; during EC-specific lineage differentiation, CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of CD34+ cells using small molecules targeting key epigenetic repressive marks may recapitulate their cardiomyogenic potential. Human CD34+ EPCs were treated with inhibitors of histone deacetylases (valproic acid) for 24 hours followed by an additional 24 hours with the DNA methyltransferase inhibitor (5-Azacytidine). This forty-eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific gene expression was maintained. Intra-myocardial transplantation of a sub-therapeutic dose of reprogrammed CD34+ cells in an acute myocardial infarction mouse model showed significant improvement in LV function compared to the same number of control CD34+ cells that are therapeutically equivalent to no treatment at all. This was histologically supported by de novo CMC differentiation. In addition to increased cardiomyogenic plasticity, drug treatment also enhanced the inherent therapeutic capacity of the CD34+ cells as shown by reduced fibrosis, increased capillary density, increased proliferation, increased cell survival and increased secretion of angiogenic factors. Taken together, our results suggest that epigenetically reprogrammed CD34+ cells are “super-CD34+ cells” that have an enhanced paracrine effect, display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.

In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations.

Abstract 199: Adipose-Derived Stem Cells from Diabetic Patients Undergo Endothelial Differentiation for Use in the Creation of a Tissue-Engineered Vascular Graft

INTRODUCTION: Surgical bypass and dialysis access creation are common procedures performed in diabetic patients. Only 55% of patients have autologous vascular tissue available for use as a conduit in these operations. We have developed an alternative conduit composed of a vascular scaffold seeded with autologous adipose-derived stem cells (ASC) differentiated into endothelial cells (EC) to create a non-thrombogenic lumen. OBJECTIVE: To examine ASC from diabetic (DM) versus non-diabetic (NDM) patients in terms of isolation efficiency, proliferation, commitment towards EC lineage, and seeding onto a vascular scaffold. METHODS: ASC were isolated from peri-umbilical liposuction specimens by collagenase dispersion (DM n=53; NDM n=145). Isolation efficiency was defined by number of ASC isolated per gram of adipose tissue. Proliferation was assessed by constructing growth curves over 14d. ASC were differentiated in endothelial growth medium for 3wk. Differentiation was determined by measuring EC-specific gene expression (CD31, vWF) using qPCR. Lastly, ASC were seeded onto decellularized vein, flow conditioned from 0-9 dynes over 5d, and examined using confocal microscopy. RESULTS: ASC isolation efficiency did not differ between DM and NDM patients (224,028 cells/gm vs. 259,345 cells/gm, respectively; P=0.21). The growth curves for DM (n=6) and NDM (n=6) ASC also appeared similar, with no significant differences observed in cells counts between days 1-14. After 1, 2, and 3wk in culture, no significant difference in CD31 or vWF expression between DM (n=6) and NDM (n=6) cells was observed. Finally, retention of ASC to vascular scaffolds under physiological shear stress was similar between the two groups. CONCLUSIONS: ASC are isolated from DM in quantities similar to those observed in NDM. These cells grow at rates similar to NDM ASC, exhibit differentiation into an EC phenotype, and line the lumen of a tissue engineered vascular graft. These results suggest that adipose tissue may prove to be a more reliable source of autologous stem cells than blood or bone marrow, where the presence of diabetes and other co-morbidities significantly decrease the availability of stem cells for use in regenerative therapies.

MAPK/ERK signalling mediates VEGF‐induced bone marrow stem cell differentiation into endothelial cell

Multi-potent adult progenitor cells (MAPCs) differentiate into endothelial cells (ECs) in the presence of vascular endothelial growth factor (VEGF). The mechanism(s) of VEGF-induced differentiation of MAPCs to ECs are not yet known. We, therefore, examined the role of mitogen-activated protein kinase/extracellular signal-regulated kinase (p42/44-MAPK/ERK1/2) signalling in endothelial differentiation from bone marrow stem cells. We observed that VEGF stimulation of MAPCs for 14 days results in a significant expression of endothelial-specific gene and/or proteins including von Willebrand factor (vWF), vascular endothelial-cadherin (VE-cadherin), VEGF receptor-2 (VEGFR2), and CD31. Up-regulation of EC-specific markers was accompanied by a cobblestone morphology, expression of endothelial nitric oxide synthase (eNOS), and Dil-Ac-LDL uptake, typical for EC morphology and function. VEGF induced a sustained activation of p42 MAPK/ERK, but not that of p44 MAPK/ERK during the course of MAPCs differentiation in a time-dependent manner up to 14 days. VEGF-induced activation of p42 MAPK/ERK also led to the nuclear translocation of MAPK/ERK1/2. Incubation of MAPCs with MAPK/ERK1/2 phosphorylation inhibitor PD98059 blocked the sustained VEGF-induced MAPK/ERK1/2 phosphorylation as well as its nuclear translocation in the differentiating MAPCs. Inhibition of MAPK/ERK1/2 phosphorylation by PD98059 also blocked the expression of EC-specific genes in these cells and their differentiation to ECs. These data suggest that VEGF induces MAPC differentiation into EC via a. MAPK/ERK1/2 signalling pathway-mediated mechanism in vitro.

High level of endothelial cell-specific gene expression by a combination of the 5′ flanking region and the 5′ half of the first intron of the VE-cadherin gene

AbstractTo develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5′ flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5′ half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin+ progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation. (Blood. 2005; 105:4657-4663)

Molecular Profiling of Angiogenic Markers: A Step Towards Interpretive Analysis of a Complex Biological Function

Gene expression profiling is now being used routinely to define complex biological events. The profiling of a large array of genes expressed in the progression of a biological response opens the door to our understanding the unique relationships between genes and their functions. Angiogenesis, the process of new blood vessel development, is a necessary component of both normal and pathological physiology. In this issue of The American Journal of Pathology, Shih and colleagues 1 have used quantitative molecular profiling of angiogenic-related factors to define some of the elements required for angiogenic profiling. Although presented as a technical advance, the basic concept of this work is that the use of quantitative molecular profiling of gene expression gives additional insight into functional interrelationships between the genes expressed during the angiogenic process. This approach can be applied in large array format as a diagnostic tool for experimental systems and pathological samples.