Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone

Abstract

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BK_Ca) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BK_Ca channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O₂. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BK_Ca channels, are promising targets. Specifically, BK_Ca channel agonists like NS1619 maintain DA patency even in the presence of O₂ and may be clinically useful.

the ductus arteriosus (da) is a fetal vessel connecting the pulmonary and systemic circulations to divert blood flow away from the uninflated lungs. At birth, increasing partial pressure of oxygen, a decrease in circulating prostaglandin E2 (PGE₂), and accompanying changes in hemodynamic forces cause the DA to constrict and functionally close (3, 11, 15, 24). In some cases, the DA fails to close resulting in a persistent left-to-right shunt, termed a patent ductus arteriosus (PDA). This condition may lead to a variety of complications including pulmonary hemorrhage, parenchymal lung disease, necrotizing enterocolitis, intraventricular hemorrhage, and death (2, 12, 40, 53, 64). Current pharmacological treatments for a PDA include indomethacin or ibuprofen, cyclooxygenase inhibitors that suppress PGE₂ (34). However, these treatments are not ideal as they do not specifically target the DA and can result in constriction of other vascular beds (10). Furthermore, despite pharmacological treatment, the DA fails to close in some infants and surgical ligation is required, a procedure accompanied by concerning surgical and anesthetic risks (52).

Conversely, neonates who have certain ductus-dependent cardiovascular lesions require a patent DA for adequate peripheral perfusion until corrective surgery can be performed (7). Currently, the only therapy for maintaining DA patency is infusion of prostaglandin E1. Unfortunately, numerous severe side effects have been reported (43, 61), reinforcing the need to develop additional therapeutic options.

Given the importance of proper DA function and the limited pharmacological modifiers currently available, it is necessary to distinguish the DA from other vessels on a molecular level in an effort to generate more specific and efficient therapies. To this end, we performed a microarray analysis to compare the genetic profiles of the fetal mouse DA to the adjacent ascending aortic (AO). Significant differences in gene expression patterns were observed. Previously, microarray analysis was used to confirm the importance of prostaglandins and oxygen-sensitive molecules like endothelin-1 in DA closure (17, 25, 33). In the current study, we have identified a subset of ion channel genes that are differentially expressed between DA and AO vessels and important for regulating vascular tone.

Ion channels, located in endothelial cells and vascular smooth muscle, integrate a constant stream of vasoconstrictor and vasodilator stimuli (29). Endothelial cells are essential mediators of tone, blood clotting, inflammation, and angiogenesis. Although endothelial cells are not excitable, they express a surprising number and variety of ion channels (51). Regulation of K⁺ and Ca²⁺ is critical for endothelial cell function. K⁺ channels set the resting membrane potential and thereby regulate Ca²⁺ flow (45, 46). In turn, Ca²⁺ flux directs endothelial production and release of many vasoactive compounds. Similarly, vascular smooth muscle cells also express multiple types of ion channels in order to regulate resting membrane potential, intracellular Ca²⁺ concentrations, and cell contractility (29, 35).

Ca²⁺-activated K⁺ (K_Ca) channels respond to changes in intracellular Ca²⁺ and govern a range of physiological activities in neurological and muscle tissues (21, 77). This family of ion channels is divided into three groups based on conductance. Small and intermediate conductance channels are preferentially expressed in endothelial cells where they respond to calcium influx and activate the synthesis of nitric oxide (8, 27). Large-conductance K_Ca (BK_Ca) channels are the most abundant channels in vascular smooth muscle. These channels are made up of a singular pore-forming alpha subunit and one of four modulatory beta subunits. Isoforms of the beta subunit are expressed in various vascular beds to confer a layer of specificity and differential sensitivity to Ca²⁺ (18). When activated, they allow for K⁺ efflux and membrane hyperpolarization, which in turn causes voltage-gated Ca²⁺ channels to close, leading to decreased intracellular Ca²⁺ and subsequent muscle relaxation (vasodilation). Conversely, when BK_Ca channels are closed, activated voltage-gated Ca²⁺ channels promote intracellular Ca²⁺ accumulation and subsequent vasoconstriction (27).

Our microarray indicates that genes encoding BK_Ca channels are expressed at significantly higher levels in the DA compared with the AO. Furthermore functional myography studies demonstrate that BK_Ca channels are physiologically active in intact DA vessels. These data provide evidence that the DA has a unique molecular signature that can distinguish it from neighboring vessels. Importantly, this study provides foundational evidence that ion channels are promising targets for novel and specific PDA therapies.

METHODS

Mice.

CD1 mice from an outbred genetic background were maintained in accordance with protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee. Timed matings were performed to generate embryos at specific stages of development. The neural crest lineage marker mouse strain Wnt1-Cre;R26R^YFP was used to illustrate a common neural crest-derived origin for DA and ascending AO smooth muscle cells. Outflow tracts from embryonic day 19 (d19) Wnt1-Cre;R26R^YFP mice were isolated and photographed in bright-field and with a yellow fluorescent protein (YFP) filter to demonstrate lineage labeled neural crest-derived cells.

Microarray analysis.

Total RNA was isolated from DA and AO vessels from d19 CD1 mice. DA and AO vessels were collected from four litters, and samples from each litter were pooled. The ascending AO was used for comparison because like the DA, its smooth muscle cells are neural crest derived (Fig. 1, A–C). Whole transcriptome expression analysis was performed using Affymetrix mouse U430 2.0 arrays. RNA quality was assessed using an Agilent 2100 BioAnalyzer before further processing and hybridization in the Vanderbilt Functional Shared Resource (http://array.mc.vanderbilt.edu/vicc/). Microarray images were scanned with an Affymetrix high resolution GenePix 4000B scanner. Raw .CEL files were subsequently uploaded into Partek Genomics Suite version 6.6 (Partek Incorporated) and processed using Robust Multi-chip Average normalization (4, 28). Partek was used to perform pairwise comparisons of average group values and one-way ANOVA with Benjamini and Hochberg (B-H) multiple hypothesis correction for analysis of DA and AO samples. Only probes that resulted in a fold-difference of at least 1.5 and B-H corrected P value of < 0.05 were considered significantly altered. Statistical analyses (including B-H correction for multiple hypothesis testing) for identification of overrepresented ontologies, functions, and pathways were performed using DAVID (http://david.abcc.ncifcrf.gov), after initial statistical data analysis was performed to identify relevant gene sets. All microarray data have been deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE51664.

Fig. 1.

Genetic profiling reveals significant differences in gene expression in the ductus arteriosus (DA) and ascending aorta (AO). A–C: lineage labeled Wnt1-Cre;R26R^YFP outflow tract under bright-field illumination (A) and a yellow fluorescent protein (YFP) filter (B) demonstrated a common neural crest-derived origin of smooth muscle cells in the AO and DA. AO and DA samples were isolated from the regions indicated in C and used for microarray and PCR analysis. D: hierarchical clustering of 5,137 probes detected as significantly different between the DA and AO. Values shown are log base 2. Red, blue, and gray indicate the highest, lowest, and median normalized signal values, respectively. Vertical dendrograms represent the individual samples, of which there are 4 replicates for each vessel type. E: principal component (PC) analysis separated samples by vessel type (red = DA, blue = AO). Circles indicate the ellipsoids (± SD of 2) for each group. The x-axis (68.3%), y-axis (13.5%), and z-axis (9.5%) components represent the total variability between experimental replicates. aAO, ascending aorta; dAO, descending aorta.

Quantitative real-time RT-PCR.

RNA was isolated from CD1 mouse vessels (d15, d17, d19, or postnatal day 1) with Trizol and the RNeasy Plus Mini Kit (Qiagen). DA and AO samples were collected from four litters at each time point, and vessels from each litter were pooled (n = 4). We generated 100 ng cDNA template using the Superscript III First Strand cDNA Synthesis kit (Invitrogen). Relative levels of gene expression were determined using SYBR-Green based quantitative RT-PCR on an iCycler iQ5 platform (Bio-Rad). Primers for each gene that was analyzed can be found in Table 1. The housekeeping gene ribosomal protein L7 (Rpl7) was used as an internal control. Triplicate ΔΔCT values were generated for each assay. The fold change in expression was determined by dividing the DA expression value by the AO expression value, which was then set to 1.

Table 1.

Primers used for real-time RT-PCR gene expression studies

Gene	Primer Sequence
Rpl7	5′-tcaatggagtaagcccaaag-3′
	5′-caagagaccgagcaatcaag-3′
Kcnma1	5′-cccagccagtgtacaaaaaggt-3′
	5′-tcaggtctgtcggtacaagttca-3′
Kcnmb1	5′-aagagctggagggcaggaa-3′
	5′-tgcccacagctgatacattga-3′
Kcnmb2	5′-gctcgtacatgcagagtgtatgg-3′
	5′-cagttaaatgtttctgtgattgacaca-3′
Clca2	5′-tcacatgagtcagagcacagc-3′
	5′-gtgtggttttgtttctttgtgc-3′
Cxcr4	5′-ggctgtagagcgagtgttgc-3′
	5′-cttcttctggtaacccatgacc-3′
Foxf1a	5′-cctcctacatcaagcaacagc-3′
	5′-aactcctttctgtcacacatgc-3′
Grik2	5′-gttagtgccaccataccatcc-3′
	5′-ccttctgtcgttaaatgtgtgc-3′
Slit2	5′-tcaatgccttctcctatagttgc-3′
	5′-agcatgaattagtccaacatgg-3′
Tcfap2b	5′-tccgagtatttgaacagacagc-3′
	5′-ccttgtcgccagttttactacc-3′
Tgfbi	5′-aaaacaatgtagtgagtgtcaataagg-3′
	5′-ctcatctcctcgttcttgtgg-3′

In situ hybridization.

In situ hybridization was performed as previously reported (56). Briefly, Kcnma1 sense and antisense ³⁵S-labeled cRNA probes were generated. P1 mice were snap-frozen and cut into 11 μm sections and mounted on glass slides. Sections were fixed with 4% paraformaldehyde/PBS, acetylated, and hybridized at 45°C for 4 h in hybridization buffer containing the ³⁵S-labeled probes. After hybridization, sections were incubated with RNaseA (20 μg/ml) at 37°C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak). Parallel sections were hybridized with sense cRNA probes to serve as negative controls. Slides were developed after 3 to 5 wk exposure periods and briefly poststained with hematoxylin and eosin.

Myography studies.

Fetal (d19) CD1 mouse DAs were isolated and mounted in microvessel perfusion chambers as previously described (55). Chambers were placed on inverted microscopes equipped with a digital image capture system (IonOptix) to record intraluminal diameters. Vessels were pressurized using a column of Krebs buffer and allowed to equilibrate, after which the pressure was increased in a stepwise manner to newborn mouse physiological mean arterial pressure (20 mmHg). Vessels were then challenged with two doses of 50 mM KCl in Krebs buffer to test for reactivity and determine maximum constriction values. Vessels that failed to constrict were excluded from further study. For dose response studies, after wash out with Krebs buffer, vessels were challenged with increasing concentrations (10⁻¹⁰ M to 10⁻³ M) of tetraethylammonium (TEA) (Acros Organics) or NS1619 (Sigma). TEA is commonly used as a BK_Ca channel inhibitor, although at concentrations that exceed the maximum dose used in this study (>5 × 10⁻³ M), it functions as a nonselective K⁺ channel blocker (63). NS1619 is a selective BK_Ca channel agonist. Following each dose, vessel diameters were allowed to plateau before the next dose was added. In other studies, vessels were treated with 10⁻⁵ M sodium nitroprusside (Sigma). After wash out with Krebs buffer, vessels were pretreated with 10⁻⁴ M TEA followed by treatment with 10⁻⁵ M sodium nitroprusside. In complementary studies, vessels were preconstricted with 12% O₂ bubbled in Krebs buffer prior to treatment with sodium nitroprusside ± TEA. For other studies, vessels were challenged with 12% O₂ bubbled in Krebs buffer. Following wash out with Krebs buffer, vessels were pretreated with 10⁻⁵ M NS1619 followed by exposure to 12% O₂. In addition, some vessels were preconstricted with 12% O₂ and then treated with 10⁻⁵ M or 10⁻⁴ M doses of NS1619. To conclude all experiments, vessels were challenged with a final dose of 50 mM KCl in Krebs buffer to demonstrate viability. Values for n indicate representative individual DAs from at least three different litters.

Statistical analysis.

For illustration purposes, changes in DA diameter were plotted as percent change in lumen diameter compared with baseline diameter at resting tone. For analytical purposes, dose response curves were modeled using unadjusted measurements of vessel diameter made at eight concentrations ranging from 10⁻¹⁰ M to 10⁻³ M. Statistical comparisons of repeated measurements of vessel diameter were analyzed with a linear mixed effects regression model. In addition to controlling for baseline vessel diameter, we included a random intercept in each mixed effects model to account for correlation arising from measuring the diameter of the same vessel at multiple concentrations. This approach allowed the comparison of intercepts and slopes separately and simultaneously with a single P value. Regression models were fit using Systat statistical software. A paired t-test was used to determine statistical significance when two conditions were compared (Figs. 3A, 4B, 4C, 6B). A P value < 0.05 was considered significant. For studies across gestational time points (Fig. 3, C and D) or multiple conditions (Fig. 6C), statistical significance was determined by performing ANOVA and Bonferroni post hoc analysis. Error bars represent standard error of the mean.

Fig. 3.

Large-conductance calcium-activated potassium (BK_Ca) channel subunits are enriched in the DA.A: quantitative real-time RT-PCR was used measure Kcnma1, Kcnmb1, and Kcnmb2 transcript levels in embryonic day (d) 19 mouse DA and AO samples (n = 4). The level of gene expression in AO samples was set to 1, and fold changes were calculated for DA samples. *Statistically significant (P < 0.001) differences in gene expression when comparing DA and AO levels. B: radioactive in situ hybridization was used to visualize Kcnma1 expression in sectioned P1 mice. An adjacent slide was hybridized using a negative control sense probe. Kcnma1 is enriched in the DA (yellow). Green indicates eosin counterstaining. C: real-time RT-PCR was used to measure Kcnma1 transcripts in the DA at different developmental stages (n = 4, *P < 0.01 compared with d15). D: real-time RT-PCR was used to measure Kcnmb1 transcripts in the DA at different developmental stages (n = 4, *P < 0.05 compared with d15). Expression levels at d15 were set to 1, and fold changes were calculated for subsequent time points. Error bars indicate SE. MPA, main pulmonary artery.

Fig. 4.

Treatment with tetraethylammonium (TEA) causes DA constriction. A: DA diameter was measured in response to increasing concentrations of TEA (10⁻¹⁰–10⁻³ M). Values represent the percent change from baseline (BL) diameter. There was a significant decrease in DA diameter following increasing doses of TEA (n = 10, P = 0.002). B: DAs treated with sodium nitroprusside (SNP, 10⁻⁵ M) dilated ∼50% from BL. Pretreatment with TEA (10⁻⁴ M) could not prevent SNP-induced dilation (n = 6). C: DAs were preconstricted with 12% O₂ (n = 8). In the presence of O₂, SNP elicited ∼114% reversal of O₂-induced constriction. Pretreatment with TEA was not able to significantly alter SNP-induced dilation (∼102% reversal). Error bars indicate SE.

Fig. 6.

Treatment with NS1619 can prevent and attenuate O₂-induced vasoconstriction. A: changes in DA diameter in response to O₂ and NS1619 were measured by pressure myography. A representative tracing is shown here. Quantification is shown in B (n = 9), illustrating exposure to 12% O₂ induced vasoconstriction that could be significantly reduced by pretreatment with 10⁻⁵ M NS1619 (P < 0.05 compared with 12% O₂ measurement). C: additional DAs (n = 5) were preconstricted with O₂ and then exposed to two doses of NS1619. 10⁻⁴ M NS1619 was able to reverse O₂-induced constriction (*P < 0.05 compared with 12% O₂ measurement). Error bars indicate SE.

RESULTS

The DA has a unique gene expression profile.

To probe for genetic variations that would reveal fundamental differences in vessel type, we performed a microarray analysis on the DA and AO isolated from d19 mouse embryos. The ascending AO was used for comparison because it is adjacent to the DA and smooth muscle cells from both vessels share a common embryonic origin, neural crest cells (Fig. 1, A–C) (32, 39, 54). Statistical analysis revealed 5,137 probes representing 4,037 unique transcripts were differentially expressed between the two vessel types. Hierarchical clustering of normalized hybridization signals for these probes successfully separated the two vessels based on gene expression patterns (Fig. 1D). Principal components analysis (PCA) of the entire expression set demonstrated that three components were sufficient to describe 65.4% of the variability between the two vessel types. PCA of only the 5,137 differentially expressed probes more successfully distinguished DA from AO samples, with the first three components sufficient to describe 91.3% of the variability between sample groups (Fig. 1E). A list of representative genes that were differentially expressed between the two vessels can be found in Supplemental Table S1.¹ Real-time RT-PCR was used to validate the expression of several of these transcripts representing different functional categories (Fig. 2).

Fig. 2.

Examples of genes upregulated in the DA. Quantitative real-time RT-PCR was used measure transcript levels in DA and AO samples (n = 4). The level of expression in AO samples was set to 1, and fold changes were calculated for DA samples. Error bars represent SE.

Of the 4,037 differentially expressed transcripts, 1,808 genes were enriched in the DA compared with the AO. Based on gene ontological analysis, the most overrepresented biological functions in this data set included focal adhesion, MAPK signaling, calcium signaling, and blood vessel morphogenesis (Table 2). Conversely, 2,229 genes were significantly downregulated in the DA samples compared with their AO counterparts. Significantly enriched biological function categories included muscle cell differentiation, regulation of blood vessel size, tissue morphogenesis, regulation of transcription, and vasodilation (Table 2). From this analysis, it is clear that although these vessels are adjacent to one another and share a common embryonic origin, they are fundamentally distinct.

Table 2.

Gene ontological analysis of overrepresented and underrepresented genes in the DA compared with the AO

Gene Ontological Category
Biological Process	Genes, n	B-H P Value
Upregulated in DA (1,808 genes)
Vascular smooth muscle contraction	19	16.7E-02
Focal adhesion	36	8.0E-03
MAPK signaling pathway	37	8.3E-02
Calcium signaling pathway	25	32.4E-02
Embryonic skeletal system development	32	2.2E-08
Embryonic skeletal system morphogenesis	26	2.8E-07
Anterior/posterior pattern formation	37	1.2E-04
Positive regulation of macromolecule biosynthetic process	88	1.7E-04
Pattern specification process	55	2.1E-04
Branching morphogenesis of a tube	26	2.7E-04
Cartilage development	23	3.7E-04
Positive regulation of cell proliferation	52	1.2E-03
Blood vessel morphogenesis	39	2.7E-03
ECM-receptor interaction	16	9.4E-02
mTOR signaling pathway	10	30.1E-02
Downregulated in DA (2,229 genes)
Muscle cell differentiation	28	3.6E-02
Tissue morphogenesis	47	4.2E-02
Regulation of blood vessel size	13	22.7E-02
Pathways in cancer	67	1.0E-03
Vasodilation	7	68.7E-02
Focal adhesion	44	5.4E-03
Endocytosis	41	4.8E-02
Cell adhesion	112	7.3E-05
Biological adhesion	112	5.3E-05
Vascular development	57	1.6E-03
Small GTPase mediated signal transduction	57	2.4E-03
Ras protein signal transduction	21	2.5E-03
Extracellular matrix organization	28	7.1E-03
Negative regulation of cell proliferation	47	2.1E-02
Negative regulator of Wnt Receptor signaling pathway	11	3.6E-02

DA, ductus arteriosus; AO, ascending aorta; B-H, Benjamini-Hochberg.

BK_Ca channels are enriched in the DA.

Among the transcripts that were enriched in the DA were subsets of genes known to be involved in vascular smooth muscle contraction and ion channel-related genes (Tables 3 and 4). Included in both groups were Kcnma1 and Kcnmb1, encoding the alpha and beta1 BK_Ca channel subunits, respectively. Real-time RT-PCR and in situ hybridizations were used to confirm the localization and enrichment (up to 2.3-fold) of the alpha subunit, Kcnma1, in the DA (Fig. 3, A and B). The beta subunit, Kcnmb1, was also upregulated (2.5-fold) in the DA compared with the AO (Fig. 3A). Another beta subunit isoform, Kcnmb2, was not detected in the microarray analysis, and real-time RT-PCR analysis revealed that this isoform was expressed at an equally low level in both vessels, demonstrating the specificity of the beta1 subunit. In addition, we determined that the expression of both Kcnma1 and Kcnmb1 in the DA is dynamic over the course of development. The peak expression of Kcnma1 was observed at postnatal day 1 (Fig. 3C), while Kcnmb1 expression was highest at d19 (Fig. 3D).

Table 3.

Genes involved in vascular smooth muscle contraction that are enriched (P value < 0.05, fold change > 1.5) in the DA

		DA/AO
Gene Name	Gene Symbol	P Value	Fold-difference
Kcnma1	potassium large conductance calcium-activated channel, subfamily M, alpha member 1	1.3E-02	2.1
Braf	Braf transforming gene	1.7E-02	1.5
Acta2	actin, alpha 2, smooth muscle, aorta	2.1E-02	2.5
Kcnmb1	potassium large conductance calcium-activated channel, subfamily M, beta member	1.6E-03	1.9
Actg2	actin, gamma 2, smooth muscle, enteric	1.3E-04	7.7
Agtr1b	angiotensin II receptor, type 1b	2.3E-02	2.0
Myh11	myosin, heavy polypeptide 11, smooth muscle	4.8E-03	2.0
Prkacb	protein kinase, cAMP dependent, catalytic, beta	1.0E-02	1.5
Calm2	calmodulin 2	5.1E-03	1.5
Ppp1r14a	protein phosphatase 1, regulatory (inhibitor) subunit 14A	2.5E-03	2.3
Mylk	myosin, light polypeptide kinase	1.4E-02	2.4

Table 4.

Ion channel-related genes differentially expressed (P value < 0.05, absolute fold change > 1.5) in the DA

		DA/AO
Gene Symbol	Gene Name	P Value	Fold-difference
Grik2	glutamate receptor, ionotropic, kainate 2 (beta 2)	2.2E-04	18.3
Trpm3	transient receptor potential cation channel, subfamily M, member 3	3.8E-04	8.9
Kcnj8	potassium inwardly-rectifying channel, subfamily J, member 8	3.6E-05	8.7
Gabra1	gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1	5.4E-04	6.9
Clca2	chloride channel calcium activated 2	3.6E-03	6.6
Gabrb2	gamma-aminobutyric acid (GABA) A receptor, subunit beta 2	1.8E-04	5.8
Clca1	chloride channel calcium activated 1	4.9E-03	4.2
Chrna7	cholinergic receptor, nicotinic, alpha polypeptide 7	1.4E-03	3.9
Gja3	gap junction protein, alpha 3	1.8E-02	3.1
Abcc9	ATP-binding cassette, sub-family C (CFTR/MRP), member 9	1.7E-02	2.8
Kcnk1	potassium channel, subfamily K, member 1	5.9E-03	2.8
Kcnk3	potassium channel, subfamily K, member 3	2.7E-02	2.8
P2r×7	purinergic receptor P2X, ligand-gated ion channel, 7	2.8E-02	2.3
Kcna4	potassium voltage-gated channel, shaker-related subfamily, member 4	4.2E-02	2.1
Kcnma1	potassium large conductance calcium-activated channel, subfamily M, alpha member 1	1.3E-02	2.1
Kcnb1	potassium voltage gated channel, Shab-related subfamily, member 1	4.1E-02	2.0
Kctd15	potassium channel tetramerisation domain containing 15	1.7E-02	2.0
Kcnmb1	potassium large conductance calcium-activated channel, subfamily M, beta member 1	1.6E-03	1.9
Clca5	chloride channel calcium activated 5	1.5E-02	1.7
Ryr3	ryanodine receptor 3	4.0E-02	1.7
Adrb2	adrenergic receptor, beta 2	4.2E-02	1.6
Clic6	chloride intracellular channel 6	5.0E-02	1.6
Kctd1	potassium channel tetramerisation domain containing 1	1.1E-02	1.6
Nalcn	sodium leak channel, nonselective	7.1E-03	1.6
Slc22a21	solute carrier family 22 (organic cation transporter), member 21	2.0E-02	1.6
Slc22a4	solute carrier family 22 (organic cation transporter), member 4	6.8E-03	1.6
Cybb	cytochrome b-245, beta polypeptide	3.2E-02	1.5
Gria1	glutamate receptor, ionotropic, AMPA1 (alpha 1)	5.7E-02	1.5
Grin3a	glutamate receptor ionotropic, NMDA3A	5.1E-02	1.5
Grm3	glutamate receptor, metabotropic 3	1.9E-02	1.5
Slc22a14	solute carrier family 22 (organic cation transporter), member 14	2.7E-02	1.5
Slc24a4	solute carrier family 24 (sodium/potassium/calcium exchanger), member 4	3.4E-03	1.5
Slc6a17	solute carrier family 6 (neurotransmitter transporter), member 17	3.7E-02	1.5
Kcne4	potassium voltage-gated channel, Isk-related subfamily, gene 4	2.3E-02	1.5
Kcnn4	potassium intermediate/small conductance calcium-activated channel, subfamily N, member	4.3E-02	−1.5
Trpm4	transient receptor potential cation channel, subfamily M, member 4	2.8E-03	−1.5
Clca3	chloride channel calcium activated 3	1.3E-02	−1.6
Clcn3	chloride channel 3	3.3E-02	−1.6
Kcnj15	potassium inwardly-rectifying channel, subfamily J, member 15	2.8E-02	−1.6
Kcnk5	potassium channel, subfamily K, member 5	8.2E-03	−1.6
Slc1a4	solute carrier family 1 (glutamate/neutral amino acid transporter), member 4	2.6E-02	−1.6
Slc4a4	solute carrier family 4 (anion exchanger), member 4	4.5E-03	−1.7
Ryr2	ryanodine receptor 2, cardiac	6.5E-03	−1.7
Scn11a	sodium channel, voltage-gated, type XI, alpha	4.0E-02	−1.7
Clic5	chloride intracellular channel 5	3.7E-02	−1.8
Kcnb1	potassium voltage gated channel, Shab-related subfamily, member 1	4.1E-03	−1.8
Tpcn2	two pore segment channel 2	2.1E-02	−1.8
Kcne3	potassium voltage-gated channel, Isk-related subfamily, gene 3	3.2E-02	−2.0
Itpr3	inositol 1,4,5-triphosphate receptor 3	1.0E-02	−2.1
Grid2	glutamate receptor, ionotropic, delta 2	4.6E-02	−2.1
Fxyd3	FXYD domain-containing ion transport regulator 3	9.0E-03	−2.2
Tmem37	transmembrane protein 37	2.7E-02	−2.2
Accn2	amiloride-sensitive cation channel 2, neuronal	2.4E-02	−2.3
Ano1	anoctamin 1, calcium activated chloride channel	1.0E-02	−2.3
Kcnd3	potassium voltage-gated channel, Shal-related family, member 3	1.5E-03	−2.3
P2rx5	purinergic receptor P2X, ligand-gated ion channel, 5	1.4E-02	−3.2
Slc39a8	solute carrier family 39 (metal ion transporter), member 8	2.0E-02	−3.2
Slco2a1	Solute carrier organic anion transporter family, member 2a1	2.0E-02	−3.9
Kcnab1	potassium voltage-gated channel, shaker-related subfamily, beta member 1	5.0E-04	−4.6
Clic3	chloride intracellular channel 3	8.0E-03	−5.8
Cacna2d3	calcium channel, voltage-dependent, alpha2/delta subunit 3	3.7E-04	−6.2

Inhibition of BK_Ca channels causes DA constriction.

To determine the functional significance of BK_Ca channels in the DA, vessel myography experiments were performed using a BK_Ca channel antagonist and agonist. Vessels (d19) were isolated and treated with increasing concentrations of the BK_Ca channel antagonist, TEA. The DA showed a modest concentration-dependent constriction after treatment with this antagonist (Fig. 4A). We next sought to determine if TEA could mitigate the effects of sodium nitroprusside (SNP), a nitric oxide donor and potent vasodilator. Vessels were first treated with SNP (10⁻⁵ M) followed by a Krebs buffer washout. Then, vessels were pretreated with TEA (10⁻⁴ M) followed by SNP administration. The luminal diameter of vessels exposed to TEA prior to SNP treatment did not significantly vary from the diameter of vessels treated with SNP alone (Fig. 4B). In a similar set of experiments, vessels were preconstricted with 12% O₂ and then exposed to SNP. After washout, vessels were then pretreated with TEA followed by SNP (Fig. 4C). In the presence of O₂, the diameter of these vessels when treated with SNP alone was not statistically different from the diameter when treated with TEA in conjunction with SNP. This suggests that blocking BK_Ca channels has no effect on nitric oxide-mediated vasodilation.

Activation of BK_Ca channels causes DA dilation.

Isolated DAs (d19) were treated with increasing concentrations of the BK_Ca channel agonist NS1619 (20). Opening BK_Ca channels caused significant vasodilation in a dose-dependent manner (Fig. 5). We then tested whether NS1619 could blunt O₂-induced DA constriction (Fig. 6, A and B). Exposure to 12% O₂ resulted in a 40–50% constriction of the DA lumen. However, pretreating the vessel with NS1619 prior to O₂ exposure caused a significant reduction in O₂-induced vasoconstriction (Fig. 6, A and B). Furthermore, NS1619 was able to reopen vessels that were preconstricted with O₂. Vessels were first constricted with 12% O₂ and then treated with two increasing doses of NS1619 (Fig. 6C). The higher dose of NS1619 (10⁻⁴ M) was able to reopen the preconstricted vessels. Taken together, these data suggest that opening BK_Ca channels is an effective means of relaxing the DA, even in the presence of O₂, the most potent regulator of DA closure.

Fig. 5.

Treatment with NS1619 causes DA dilation. DA diameter was measured in response to increasing concentrations of NS1619. Values represent the percent change from BL diameter. There was a significant increase in vessel diameter following increasing doses of NS1619 (n = 7, P < 0.001). Error bars indicate SE.

DISCUSSION

It is well known that vasculature is heterogeneous. Variations in endothelial cells (37), smooth muscle (70), and connective tissue (23, 57) have been noted along the vascular tree and are thought to give rise to regional differences in function as well as disease susceptibility (9, 42, 65, 68). The source of these variations is often attributed to the diverse embryonic origins of vasculogenic cells (54, 79). Indeed, vascular progenitor cells can arise from neural crest cells (32, 41), splanchnic mesoderm (76), somitic mesoderm (74), and mesothelial cells (60). However, differing embryonic origin is not sufficient to define all variations observed among vascular beds. The smooth muscle of the DA and ascending AO are both derived from neural crest cells, and yet they display fundamental differences in form and function.

Using microarray analysis, we have identified patterns in gene expression that differentiate the DA from the adjacent ascending AO. Some of the genes that were differentially expressed in our array analysis were expected given their known roles in DA biology. For instance, mice lacking the prostaglandin E receptor 4 (Ptger4) have prominent PDAs (50, 58, 59) and have altered expression of contractile, migration, growth, and vascular tone genes (25). In our study, Ptger4 was enriched 3.7-fold in the DA (Supplemental Table S1). Genes known to be associated with PDA including Tfap2b (80), Jag1 (22), Hpgd (16), and others were also identified in our array (Supplemental Table S1).

It was also not surprising that adhesion factors and extracellular matrix genes were enriched in the DA (Table 2). During closure of the DA, smooth muscle cells migrate into the subendothelial space causing intimal mounds to form (62). These structures occlude the lumen, remodel, and create a permanent seal. Extracellular matrix components including fibronectin, laminin, and collagens are critical for proper smooth muscle cell migration into the subendotheial space (5, 14, 78). Adhesion molecules like vascular cell adhesion molecule (Vcam-1) and integrins are also implicated in DA closure. Vcam-1 facilitates mononuclear cell adherence to the lumen, a process linked to mound formation and successful DA remodeling (72). Similarly, integrins provide an essential link between vascular cells and the extracellular matrix, providing cues for DA remodeling (13).

The unexpected prevalence of ion channel-related genes in our analysis was of particular interest (Table 4). In general, ion channels have been linked to a variety of diseases, termed channelopathies, including cystic fibrosis, epilepsy, diabetes, and osteoporosis (36). Specifically, abnormal BK_Ca channels play important roles in several diseases. A gain-of-function COOH-terminal mutation in the alpha subunit has been linked to epilepsy with paroxysmal dyskinesia (19), while loss-of-function mutations are tied to urinary incontinence, bladder hyperactivity, and erectile dysfunction (47, 75). Similarly, a decrease in the expression of the beta1 subunit has been associated with hypertension (1).

Vascular smooth muscle cells express multiple types of ion channels including K⁺ channels, Ca²⁺ channels, Cl⁻ channels, and stretch-activated cation channels (29). Examples of each of these subtypes were found to be differentially expressed between DA and AO samples. As K⁺ channels are the dominant channel type in vascular smooth muscle, it is not surprising that the majority of ion channel-related DA studies have focused on K⁺ channels (71). Tristani-Firouzi et al. (69) were the first to report that specific K⁺ channels regulate DA tone. In rabbits, they found O₂ inhibited a voltage-gated K⁺ channel (58-pS) leading to smooth muscle cell depolarization and DA constriction. Similarly, the Archer laboratory reported that in humans, K_V channels control basal DA tone and are important in the acute phase of normoxic constriction (48). Keck et al. (38) elaborated on this finding by demonstrating that pharmacologic inhibition of K_V channels (K_V 1.1, K_V 1.5, and K_V 2.1) caused accumulation of intracellular Ca²⁺ in DA smooth muscle cells. Furthermore, Nakanishi and colleagues (49) demonstrated that O₂ can also close K_ATP channels in the DA, leading to Ca²⁺ accumulation and vasoconstriction. Yet the function of BK_Ca channels in the DA has been heretofore understudied. In work done in rats, Sun and colleagues (66) showed that BK_Ca channels are present in DA smooth muscle and endothelial cells but do not contribute to O₂-induced constriction. Thebaud et al. (67) targeted BK_Ca channels in the DA endothelium in rabbits. Sildenafil was used to inhibit endothelial BK_Ca channels to oppose O₂-induced constriction. We have demonstrated that the BK_Ca agonist NS1619 can be used to maintain DA patency even in the presence of O₂. These data suggest targeting potassium channels (73) may be a way to affect DA tone with reduced off-target effects as it has been shown that BK_Ca channel inhibitors including TEA have little effect on the microvasculature (30, 31), and we have shown that BK_Ca channel genes are specifically enriched in the DA.

Importantly, our data demonstrate differences in expression of the alpha BK_Ca channel subunit, Kcnma1, and the beta subunit, Kcnmb1 (Fig. 3A). We show that the expression of both subunits increases in the DA with gestational age (Fig. 3C). While only one alpha subunit has been described, four mammalian beta subunits have been identified (Kcnmb1–4). Each of these beta subunits plays a unique role in defining channel activation/deactivation rates and regulating Ca²⁺ sensitivity (6), thereby defining the functional properties of BK_Ca channels in a cell-specific manner (73). The activating beta 1 isoform, Kcnmb1, is restricted to smooth muscle, exhibits high Ca²⁺ sensitivity and is slow to deactivate (26, 44). We found that this isoform is significantly enriched in the DA compared with the AO. In contrast, we found an equally low level of expression of the inactivating Kcnmb2 subunit in both DA and AO samples. Similarly, Sun and colleagues (66) reported high levels of Kcnmb1 expression in the rat DA, but low to undetectable levels of Kcnmb2 and Kcnmb4, a more distantly related family member that confers slower activation kinetics. These data identify a particular type of BK_Ca channel that is enriched in the DA, one that is composed primarily of Kcnmb1 subunits, suggesting elevated Ca²⁺ sensitivity and slow deactivation kinetics. Importantly, the expression of the Kcnmb1 subunit peaks at d19, precisely when the DA is poised for closure.

This study demonstrates that the DA has a unique genetic signature that can be exploited in the development of new therapies. Ion channels appear to be a rich source of DA-specific drug targets that could be used to precisely regulate DA tone without affecting other vascular beds. BK_Ca channels are particularly promising given the differences noted in channels expressed in vascular vs. nonvascular tissue and, importantly, in channels expressed in different vascular beds. It will be important to explore other DA-specific genes to discover and refine therapeutic options.

GRANTS

This work was supported by National Institutes of Health Grants HD-44741 (to B. C. Paria) and HL-77395, HL-96967, and HL-109199 (to J. Reese).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: E.L.S. and G.E. conception and design of research; E.L.S., G.E., C.H., N.B., I.W., E.R.P., and B.C.P. performed experiments; E.L.S., G.E., C.L.G., R.B.C., J.Z.S., and J.R. analyzed data; E.L.S., C.L.G., and J.R. interpreted results of experiments; E.L.S. and C.L.G. prepared figures; E.L.S. drafted manuscript; E.L.S., B.C.P., R.B.C., J.Z.S., and J.R. edited and revised manuscript; E.L.S., G.E., C.L.G., N.B., B.C.P., R.B.C., J.Z.S., and J.R. approved final version of manuscript.