Galectin-3 and aldosterone as potential tandem biomarkers in pulmonary arterial hypertension.

BACKGROUND: Several studies have identified circulating biomarkers to be associated with the presence and severity of pulmonary arterial hypertension (PAH). Recent evidence supports a role for galectin-3 (Gal-3) and the mineralcorticoid aldosterone in left ventricular failure. However, studies on aldosterone together with Gal-3 in PAH are lacking. OBJECTIVE: We investigated a novel Aldosterone-galectin-3 (Gal-3) tandem and several other potential PAH biomarkers and their association with the disease severity. METHODS: A total of 57 patients, 41 with idiopathic PAH. (IPAH) and 16 with PAH associated with connective tissue disease (CTD), and 8 age-matched, non-relative controls were studied. Gal-3, aldosterone and other potential protein plasma concentrations were measured by single ELISA and multi-array MSD (Meso Scale Discovery) technology. RESULTS: Gal-3 values were increased in both patients with IPAH (12.2±0.6 ng/mL; p<0.05) and with PAH-CTD (14.1±1.6 ng/mL; p<0.05) versus control (8.5±0.9 ng/mL), while aldosterone was significantly elevated in IPAH only (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05). In addition, aldosterone, Gal-3, and N-terminal pro-brain natriuretic peptide (NT-proBNP) values were all higher in patients in WHO functional class II-III versus PAH functional class I or controls. The vascular injury marker intercellular adhesion molecule 1 (ICAM-1) was increased in IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively), whereas vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin-12 (IL-12) were elevated in PAH-CTD only (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively). CONCLUSIONS: Heightened Gal-3 and aldosterone plasma concentrations in PAH patients indicate a role for Gal-3 signalling in the pathobiology of IPAH and PAH-CTD, and may serve as biomarkers for functional status and progression of disease.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Microfluidic capture of endothelial colony-forming cells from human adult peripheral blood: phenotypic and functional validation in vivo.

INTRODUCTION: Endothelial colony-forming cells (ECFCs) are endothelial progenitors that circulate in peripheral blood and are currently the subject of intensive investigation due to their therapeutic potential. However, in adults, ECFCs comprise a very small subset among circulating cells, which makes their isolation a challenge. MATERIALS AND METHODS: Currently, the standard method for ECFC isolation relies on the separation of mononuclear cells and erythrocyte lysis, steps that are time consuming and known to increase cell loss. Alternatively, we previously developed a novel disposable microfluidic platform containing antibody-functionalized degradable hydrogel coatings that is ideally suited for capturing low-abundance circulating cells from unprocessed blood. In this study, we reasoned that this microfluidic approach could effectively isolate rare ECFCs by virtue of their CD34 expression. RESULTS: We conducted preclinical experiments with peripheral blood from four adult volunteers and demonstrated that the actual microfluidic capture of circulating CD34(+) cells from unprocessed blood was compatible with the subsequent differentiation of these cells into ECFCs. Moreover, the ECFC yield obtained with the microfluidic system was comparable to that of the standard method. Importantly, we unequivocally validated the phenotypical and functional properties of the captured ECFCs, including the ability to form microvascular networks following transplantation into immunodeficient mice. DISCUSSION: We showed that the simplicity and versatility of our microfluidic system could be very instrumental for ECFC isolation while preserving their therapeutic potential. We anticipate our results will facilitate additional development of clinically suitable microfluidic devices by the vascular therapeutic and diagnostic industry.

Tag-free microfluidic separation of cells against multiple markers.

Conventional cell separation against multiple markers generally requires the attachment of antibody tags, typically fluorescent or magnetic, to selected cell types in a heterogeneous suspension. This work describes how such separation can be accomplished in a series of microfluidic systems without the need for such tags. Two capture stages containing antibody-functionalized alginate hydrogels are utilized for the isolation of CD34+ and Flk1+ cells from untreated, whole human blood. The capture-release capability of these degradable coatings is harnessed by a mixing chamber and a simple valving system such that the suspension emerging from the first capture stage is prepared for the second capture stage for further enrichment. With this configuration, we demonstrate the isolation of CD34+/Flk1+ endothelial progenitor cells from blood enabled by the depletion of CD34+/Flk1-hematopoietic stem cells population. This ability to achieve isolation of cells against multiple markers in an untagged separation method is of particular significance in applications involving cell implantation-based therapeutics including tissue engineering and molecular analysis.

Design and validation of an endothelial progenitor cell capture chip and its application in patients with pulmonary arterial hypertension.

The number of circulating endothelial progenitor cells (EPCs) inversely correlates with cardiovascular risk and clinical outcome, and thus has been proposed as a valuable biomarker for risk assessment, disease progression, and response to therapy. However, current strategies for isolation of these rare cells are limited to complex, laborious approaches. The goal of this study was the design and validation of a disposable microfluidic platform capable of selectively capturing and enumerating EPCs directly from human whole blood in healthy and diseased subjects, eliminating sample preprocessing. We then applied the "EPC capture chip" clinically and determined EPC numbers in blood from patients with pulmonary arterial hypertension (PAH). Blood was collected in tubes and injected into polymeric microfluidic chips containing microcolumns pre-coated with anti-CD34 antibody. Captured cells were immunofluorescently stained for the expression of stem and endothelial antigens, identified and counted. The EPC capture chip was validated with conventional flow cytometry counts (r = 0.83). The inter- and intra-day reliability of the microfluidic devices was confirmed at different time points in triplicates over 1-5 months. In a cohort of 43 patients with three forms of PAH (idiopathic/heritable, drug-induced, and connective tissue disease), EPC numbers are ≈50% lower in PAH subjects vs. matched controls and inversely related to two potential disease modifiers: body mass index and postmenopausal status. The EPC capture chip (5 × 30 × 0.05 mm(3)) requires only 200 µL of human blood and has the strong potential to serve as a rapid bedside test for the screening and monitoring of patients with PAH and other proliferative cardiovascular, pulmonary, malignant, and neurodegenerative diseases.

Engineered alginate hydrogels for effective microfluidic capture and release of endothelial progenitor cells from whole blood.

Microfluidic adhesion-based cell separation systems are of interest in clinical and biological applications where small sample volumes must be processed efficiently and rapidly. While the ability to capture rare cells from complex suspensions such as blood using microfluidic systems has been demonstrated, few methods exist for rapid and nondestructive release of the bound cells. Such detachment is critical for applications in tissue engineering and cell-based therapeutics in contrast with diagnostics wherein immunohistochemical, proteomic, and genomic analyses can be carried out by simply lysing captured cells. This paper demonstrates how the incorporation of four-arm amine-terminated poly(ethylene glycol) (PEG) molecules along with antibodies within alginate hydrogels can enhance the ability of the hydrogels to capture endothelial progenitor cells (EPCs) from whole human blood. The hydrogel coatings are applied conformally onto pillar structures within microfluidic channels and their dissolution with a chelator allows for effective recovery of EPCs following capture.