Introduction
The formation of coronary vessels is fundamental in heart development. Our lab has demonstrated that Reelin, an extracellular matrix glycoprotein, is involved in the formation of ...coronary vessels. Reelin expression is localized to the vascular endothelial cells of coronary vessels in embryonic mice. Human dermal microvascular endothelial cells (HDMECs) were used to examine the potential role of Reelin in vascular development as they appropriately model events involved in vessel formation and endogenously express RELN mRNA. Previous studies from our lab indicated that small interfering RNA (siRNA)‐mediated gene silencing of RELNin HDMECs reduced cell migration and basal cell membrane permeability, and increased capillary‐like tube formation. Based on these results, we believe Reelin is involved in vasculogenesis and angiogenesis. This prompted us to analyze expression of angiogenic transcripts by qPCR array in negative control and RELN knockdown (KD) HDMECs. Our results indicated significant upregulation of ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs), and significant changes in expression of other angiogenesis‐related transcripts.
Study Objective
This study aimed to validate the angiogenesis array data by individual TaqMan qPCR analysis of mRNA expression of the significantly altered transcripts. Also, we aimed to analyze the encoded protein expression of these transcripts in negative control and RELN KD cells. We hypothesized that significant alterations in expression of ADAMTS1 and other angiogenic transcripts leads to changes in their protein expression in our RELN KD cells and mediates Reelin signaling in human vascular endothelial cells.
Methods
To validate our angiogenesis qPCR array data, we utilized HDMECs as an in vitro system to examine mRNA and protein expression. Gene‐specific siRNAs were used to target and knockdown endogenous expression of RELN in HDMECs. Total RNA was isolated from negative control and RELNKD HDMECs and it was analyzed by qPCR with TaqMan probes for RELN, ADAMTS1, GAPDH, 18SRNA and other angiogenic transcripts. Also, protein expression of the encoded transcripts was analyzed by immunofluorescence and western blot in negative control and RELN KD HDMECs.
Results
RELN KD HDMECs exhibited a >90% reduction in Reelin protein expression compared to negative control HDMECs. ADAMTS1 mRNA was significantly upregulated in RELN KD cells while expression of other angiogenic transcripts were also altered in these cells. We demonstrated by immunofluorescence that ADAMTS1 is localized to the nucleus, and it is detected in HDMEC lysates by western blot analysis. In addition, ADAMTS1 protein expression differed between negative control and RELN KD HDMECs.
Conclusions
We conclude that ADAMTS1 mRNA is upregulated in RELN KD HDMECs versus negative control cells. Knockdown of RELN in HDMECS altered cell migration, membrane permeability, capillary‐like tube formation and expression of angiogenic transcripts. These findings provide key evidence that ADAMTS1 and other angiogenic transcripts may be novel mediators of Reelin signaling in human vascular endothelial cells.
Introduction
The epicardial layer of the heart is formed during cardiovascular development from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate out of the PE to ...surround and adhere to the myocardium to form a layer of epithelial cells that compose the epicardium. A subset of the epicardial cells delaminate and migrate into the subepicardial space where they undergo epithelial‐mesenchymal transition (EMT) to become epicardial‐derived cells (EPDCs). These EPDCs invade the myocardium and differentiate into a subset of cells that make up the coronary vessels. During this entire process, the epicardium receives input from several transcription factors including Tbx5. Mice with a conditional deletion of Tbx5 exhibit impaired epicardium formation and coronary vessel development. Tbx5‐deficient mouse hearts have a significant decrease in mRNA expression of adherens junction associated protein (Ajap1). Our studies show that knockdown of endogenous AJAP1 (AJAP1 KD) mRNA expression in epithelial cells leads to increased cell adhesion, reduced cell migration, enhanced expression of epithelial markers of EMT, and altered expression of transcripts associated with adherens junctions, tight junctions and EMT. Specifically, our qPCR array data shows a significant alteration in ZEB2 mRNA expression in AJAP1 KD cells versus negative control cells.
Study Objective
The goal of this study was to examine the role of AJAP1 in regulating protein expression of ZEB2 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZEB2 protein is an important functional mediator of AJAP1 in epithelial cells for undergoing EMT.
Methods
We utilized the primary human mammary epithelial cell line (HMEpiC) as an in vitro system to examine contributions of AJAP1 to epithelial cell biology. We silenced AJAP1 mRNA expression in HMEpiCs using small interfering RNAs and examined protein expression of ZEB2. We compared ZEB2 expression in negative control versus AJAP1 KD HMEpiCs.
Results
Reduced expression of AJAP1transcripts produced noticeable changes in protein expression of ZEB2 in epithelial cells, and it had significant effects on expression of adherens junction‐related transcripts. Western blot analysis revealed an increase in ZEB2 protein expression in AJAP1 KD versus negative control HMEpiCs.
Conclusion
Our data indicate that AJAP1 contributes to epithelial cell function, and it does this potentially by mediating ZEB2 protein expression. ZEB2 is known to regulate EMT, which is a biological process that is crucial for the formation of the epicardium and its subsequent formation of the coronary vasculature.
Diseases of the cardiovascular system that cause sudden cardiac deaths are often caused by lethal arrhythmias that originate from defects in the cardiac conduction system. Development of the cardiac ...conduction system is a complex biological process that can be wrought with problems. Although several genes involved in mature conduction system function have been identified, their association with development of specific subcomponents of the cardiac conduction system remains challenging. Several transcription factors, including homeodomain proteins and T-box proteins, are essential for cardiac conduction system morphogenesis and activation or repression of key regulatory genes. In addition, several transcription factors modify expression of genes encoding the ion channel proteins that contribute to the electrophysiological properties of the conduction system and govern contraction of the surrounding myocardium. Loss of transcriptional regulation during cardiac development has detrimental effects on cardiogenesis that may lead to arrhythmias. Human genetic mutations in some of these transcription factors have been identified and are known to cause congenital heart diseases that include cardiac conduction system malformations. In this review, we summarize the contributions of several key transcription factors to specification, patterning, maturation, and function of the cardiac conduction system. Further analysis of the molecular programs involved in this process should lead to improved diagnosis and therapy of conduction system disease.
To dissect the
TBX5
regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type
, ...CRE, Tbx5
lox/
+
and Tbx5
del/
+
mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between
TBX5
, arrhythmia and heart development.
Holt-Oram syndrome is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ impaired coronary blood vessel formation. ...However, the potential activity of Tbx5 in the epicardium itself, and the role of Tbx5 in mammalian coronary vasculogenesis, remains largely unknown.
To evaluate the consequences of altered Tbx5 gene dosage during proepicardial organ and epicardial development in the embryonic chick and mouse.
Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick proepicardial organ and proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/)) impaired normal proepicardial organ cell development, inhibited epicardial and coronary blood vessel formation, and altered developmental gene expression. The generation of epicardial-derived cells and their migration into the myocardium were impaired between embryonic day (E) 13.5 to 15.5 in mutant hearts because of delayed epicardial attachment to the myocardium and subepicardial accumulation of epicardial-derived cells. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wild-type hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1α upregulation and increased binding of hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity when compared with wild-type mice.
Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and in mouse coronary vascular formation.
Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond ...phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding.
Previously, we identified an R674Q missense mutation in the MYH8 gene encoding the myosin heavy chain perinatal isoform (MyHCpn) in individuals with a rare CNC variant who exhibit typical CNC ...findings along with the trismus pseudocamptodactyly limb contracture syndrome.
Thoracic aortic aneurysm (TAA) is a common progressive disorder involving gradual dilation of the ascending and/or descending thoracic aorta that eventually leads to dissection or rupture. ...Nonsydromic TAA can occur as a genetically triggered, familial disorder that is usually transmitted in a monogenic autosomal dominant fashion and is known as familial TAA. Genetic analyses of families affected with TAA have identified several chromosomal loci, and further mapping of familial TAA genes has highlighted disease-causing mutations in at least 4 genes: myosin heavy chain 11 (MYH11), α-smooth muscle actin (ACTA2), and transforming growth factor β receptors I and II (TGFβRI and TGFβRII).
We evaluated 100 probands to determine the mutation frequency in MYH11, ACTA2, TGFβRI, and TGFβRII in an unbiased population of individuals with genetically mediated TAA. In this study, 9% of patients had a mutation in one of the genes analyzed, 3% of patients had mutations in ACTA2, 3% in MYH11, 1% in TGFβRII, and no mutations were found in TGFβRI. Additionally, we identified mutations in a 75 base pair alternatively spliced TGFβRII exon, exon 1a that produces the TGFβRIIb isoform and accounted for 2% of patients with mutations. Our in vitro analyses indicate that the TGFβRIIb activating mutations alter receptor function on TGFβ2 signaling.
We propose that TGFβRIIb expression is a regulatory mechanism for TGFβ2 signal transduction. Dysregulation of the TGFβ2 signaling pathway, as a consequence of TGFβRIIb mutations, results in aortic aneurysm pathogenesis.
Introduction
Establishment of the coronary circulation is a critical step in heart development. Formation of functional coronary vessels is required for the heart to undergo successful compaction of ...the ventricular myocardium. Oxygen and nutrients are delivered to the contractile myocardium through the coronary vasculature. Molecular signaling pathways that control formation of the coronary vessels are being identified. Immunofluorescent studies from our lab indicate that the Reelin extracellular matrix glycoprotein is expressed within the vascular endothelial cells of developing and mature coronary vessels of the mouse heart. The Reelin signaling pathway is known to contribute to brain and lymphatic system development by influencing neuronal and endothelial cell behaviors such as adhesion and migration. However, its role in coronary vascular development is as yet unidentified.
Study Objective
The goal of this study was to elucidate the potential roles of Reelin in formation of the coronary vasculature by identifying how it regulates vascular endothelial cell behaviors. We hypothesized that Reelin signaling regulates establishment of coronary vessel integrity during cardiogenesis by influencing vascular endothelial cell adhesion as well as cell membrane permeability and resistance.
Methods
We utilized the primary human dermal microvascular endothelial cell line (HDMEC) as an in vitro system to examine contributions of Reelin to vascular endothelial cell behavior. A small interfering RNA‐mediated approach was used to silence the endogenous RELN gene in HDMECs and reduce Reelin protein expression. Transfected HDMECs were subjected to cell‐based assays to assess changes in vascular endothelial cell behavior in response to RELN silencing. We assessed vascular endothelial cell adhesion on extracellular matrices that are required during coronary vessel development. Vessel integrity was examined by assaying endothelial cell membrane permeability and resistance in RELN‐silenced cells. Also, we examined the effect of Reelin expression on cell viability.
Results
RELN‐silenced HDMECs exhibited a >90% reduction in Reelin expression in comparison to non‐targeting, negative control HDMECs. This significant Reelin reduction altered vascular endothelial cell adhesion to certain extracellular matrices. These RELN‐silenced HDMECs exhibited a significant decrease in baseline cell membrane permeability and concomitant changes in cell membrane resistance. Furthermore, we observed changes in cell morphology in RELN‐silenced cells versus control cells. Preliminary data suggests that loss of RELN expression affects vascular endothelial cell morphology and expression of components within the Reelin signaling pathway.
Conclusions
Based on this evidence, we conclude that Reelin influences adhesion, morphology, membrane permeability and membrane resistance of vascular endothelial cells in order to regulate vessel wall integrity. These findings may provide key evidence as to how Reelin expression in nascent vascular endothelial cells impacts formation of the coronary vessels during mammalian cardiovascular development.
Support or Funding Information
AHA 17AIREA3360773 and PCOM Center for Chronic Disorders of Aging
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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