The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth factor binding protein-2 (LTBP2) as a circulating factor associated with disease progression in fibrotic lung diseases in humans (including IPF), but its role in the development of fibrosis is incompletely defined. LTBP2 competes with the large latent transforming growth factor-beta (TGFβ) complex (LLC) for binding to the N-terminus of fibrillin and is thought to promote the release of active TGFβ. We hypothesized that LTBP2 deficiency would promote LLC sequestration in matrix and reduce TGFβ signaling. We recently reported an LTBP2 knockout (Ltbp2 -/-) mouse with no baseline lung abnormalities. Here we show that Ltbp2 -/- mice exposed to either bleomycin or silica have a significant reduction in fibrosis compared to wild type controls. Consistent with reduced fibrosis, after bleomycin Ltbp2 -/- mouse lungs have reduced TGFβ signaling and isolated fibroblasts from Ltbp2 -/- mice exhibit impaired migration in an in vitro wound closure assay. Transcriptomic analysis of bleomycin-treated control and Ltbp2 -/- mouse lung tissue identified multiple LTBP2-regulated genes, including the lncRNA antisense of IGFR2 non-coding RNA (Airn) which has reported antifibrotic effects. Interestingly, we also observed that Ltbp2 -/- mice had impaired epithelial repair after bleomycin treatment, a phenotype that also occurred in a naphthalene model of club cell injury. These findings provide evidence that LTBP2 is profibrotic and facilitates TGFβ signaling but is also required for normal airway epithelial repair.

BACKGROUND:Endothelial cells (ECs) are the primary producers of elastin in the internal elastic lamina (IEL) of resistance arteries. These arteries have distinct gaps in their IEL where ECs facilitate heterocellular communication with smooth muscle in a signaling microdomain termed the myoendothelial junction. However, the contribution of the IEL to vasodilation and blood pressure in resistance arteries is not well understood. METHODS:An endothelial-specific elastin knockout mouse (EC-specific Elnfl/fl/Cre+) was used to alter the IEL and myoendothelial junctions. Myoendothelial junction resident proteins were localized by en face, pressure myography assessed the effect of elastin depletion on vessel dilation, and blood pressure was measured using radiotelemetry. RESULTS:Using single-cell RNA-sequencing, we found Eln mRNA enriched in arterial endothelium. In EC-specific Elnfl/fl/Cre+ mice, the localization of the myoendothelial junction resident protein Hbα (α hemoglobin) becomes diffuse and disorganized. Normally, Hbα regulates eNOS (endothelial nitric oxide synthase) by sequestering NO, promoting endothelial-derived hyperpolarization as the predominant vasodilation mechanism. However, in EC-specific Elnfl/fl/Cre+ mice, Hbα expression and interaction with eNOS are significantly reduced, corresponding to increased NO signaling via acetylcholine dilation. Intact arteries also exhibit decreased smooth muscle contractility with the diminished IEL. These vascular deficiencies suggested a hypotensive phenotype, but EC-specific Elnfl/fl/Cre+ mice's blood pressure was not different from controls. CONCLUSIONS:Our findings suggest that elastin deficiency in resistance arteries alters their vasoreactive properties, resulting in poor contraction and dilation. Furthermore, the absence of the holes in the IEL mislocalizes Hbα and eNOS in resistance arteries, switching the vasodilatory mechanism from endothelial-derived hyperpolarization to NO signaling, mimicking larger conduit arteries.

Thoracic and abdominal aortic aneurysms (TAAs and AAAs, respectively) share morphological features but have distinct clinical and hereditary characteristics. Studies using bulk RNA comparisons revealed distinct patterns of gene expression in human TAA and AAA tissues. However, given the summative nature of bulk RNA studies, these findings represent the totality of gene expression without regards to the differences in cellular composition. Single-cell RNA sequencing provides an opportunity to interrogate cell-type-specific transcriptomes. Single cell RNA sequencing datasets from mouse TAA (GSE153534) and AAA (GSE164678 and GSE152583) with respective controls were obtained from the Gene Expression Omnibus. Bioinformatic analysis was performed with the Seurat 4, clusterProfiler, and Connectome software packages (V1.0.1). Immunostaining was performed with standard protocols. Within normal and aneurysmal aortae, three unique populations of cells that express smooth muscle cell (SMC) markers were identified (SMC1, SMC2, and SMCmod). A greater proportion of TAA SMCs clustered as a unique population, SMCmod, relative to the AAA SMCs (38% vs. 10–12%). These cells exhibited transcriptional features distinct from other SMCs, which were characterized by Igfbp2 and Tnfrsf11b expression. Genes upregulated in TAA SMCs were enriched for the Reactome terms “extracellular matrix organization” and “insulin-like growth factor (IGF) transport and uptake by IGF binding proteins (IGFBPs)”, indicating a role for Igfbp2 in TAA pathogenesis. Regulon analysis revealed transcription factors enriched in TAAs and AAAs. Validating these mouse bioinformatic findings, immunostaining demonstrated that both IGFBP2 and TNFRSF11B proteins increased in human TAAs compared to AAAs. These results highlight the unique cellular composition and transcriptional signature of SMCs in TAAs and AAAs. Future studies are needed to reveal the pathogenetic pathways of IGFBP2 and TNFRSF11B.

Arterial stiffening occurs during natural aging, is associated with an increased risk of adverse cardiovascular events, and can follow different timelines in males and females. One mechanism of arterial stiffening includes remodeling of the extracellular matrix (ECM), which alters the wall material properties. We used elastin haploinsufficient (Eln+/-) and wildtype (Eln+/+) mice to investigate how material properties of two different arteries (ascending aorta and carotid artery) change with age, sex, and ECM composition. We used a constitutive model by Dong and Sun that is based on the Holzapfel-Gasser-Ogden (HGO) type, but does not require a discrete number of fibrous ECM families and allows varied deformation coupling. We find that the amount of deformation coupling for the best fit model depends on the artery type. We also find that remodeling to maintain homeostatic (i.e., young, wildtype) values of biomechanical parameters with age, sex, and ECM composition depends on the artery type, with ascending aorta being more adaptable than carotid artery. Fitted material constants indicate sex-dependent remodeling that may be important for determining the time course of arterial stiffening in males and females. We correlated fitted material constants with ECM composition measured by biochemical (ascending aorta) or histological (carotid artery) methods. We show significant correlations between ECM composition and material parameters for the mean values for each group, with biochemical measurements correlating more strongly than histological measurements. Understanding how arterial stiffening depends on age, sex, ECM composition, and artery type may help design effective, personalized clinical treatment strategies.

BACKGROUND:Latent TGFβ binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFβ, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS:Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS:Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Supravalvular aortic stenosis (SVAS) is an autosomal dominant disease resulting from elastin (ELN) haploin-sufficiency. Individuals with SVAS typically develop a thickened arterial media with an increased number of elastic lamellae and smooth muscle cell (SMC) layers and stenosis superior to the aortic valve. A mouse model of SVAS (Eln+/-) was generated that recapitulates many aspects of the human disease, including increased medial SMC layers and elastic lamellae, large artery stiffness, and hypertension. The vascular changes in these mice were thought to be responsible for the hypertension phenotype. However, a renin gene (Ren) duplication in the original 129/Sv genetic background and carried through numerous strain back-crosses raised the possibility of renin-mediated effects on blood pressure. To exclude excess renin activity as a disease modifier, we utilized the Cre-LoxP system to rederive Eln hemizygous mice on a pure C57BL/6 background (Sox2-Cre;Elnf/f). Here we show that Sox2-Cre;Eln+/f mice, with a single Ren1 gene and normal renin levels, phenocopy the original global knockout line. Characteristic traits include an increased number of elastic lamellae and SMC layers, stiff elastic arteries, and systolic hypertension with widened pulse pressure. Importantly, small resistance arteries of Sox2-Cre;Eln+/f mice exhibit a significant change in endothelial cell function and hypercontractility to angiotensin II, findings that point to pathway-specific alterations in resis-tance arteries that contribute to the hypertensive phenotype. These data confirm that the cardiovascular changes, particularly systolic hypertension, seen in Eln+/-mice are due to Eln hemizygosity rather than Ren duplication.(c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid -channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

Abdominal aortic aneurysms (AAAs) represent a multifactorial, proteolytic disorder involving disintegration of the matrix structure within the AAA wall. Intrinsic deficiency of adult vascular cells to regenerate and repair the wall elastic matrix, which contributes to vessel stretch and recoil, is a major clinical challenge to therapeutic reversal of AAA growth. In this study, we investigate the involvement of epidermal growth factor receptor-mitogen activated protein kinase (EGFR-MAPK) pathway in the activation of aneurysmal smooth muscle cells (SMCs) by neutrophil elastase, and how EGFR can be targeted for elastic matrix regeneration. We have demonstrated that neutrophil elastase activates EGFR and downregulates expression level of key elastin homeostasis genes (elastin, crosslinking enzyme-lysyl oxidase, and fibulin4) between a dose range of 1-10 mu g/mL (p < 0.05). It also incites downstream proteolytic outcomes by upregulating p-extracellular signal-regulated kinase (ERK)1/2 (p < 0.0001) and matrix metalloprotease 2 (MMP2) at a protein level, which is significantly downregulated upon EGFR-specific inhibition by tyrosine kinase inhibitor AG1478 (p-ERK1/2 and MMP2 [p < 0.05]). Moreover, we have shown that EGFR inhibition suppresses collagen amounts in aneurysmal SMCs (p < 0.05) and promotes robust formation of elastic fibers by enhancing its deposition in the extracellular space. Hence, the EGFR-MAPK pathway in aneurysmal cells can be targeted to provide therapeutic effects toward stimulating vascular matrix regeneration. Impact statementProteolytic disorders such as aortal expansions, called abdominal aortic aneurysms (AAAs), are characterized by naturally irreversible enzymatic breakdown and loss of elastic fibers, a problem that has not yet been surmounted by existing tissue engineering approaches. In this work, we show, for the first time, how epidermal growth factor receptor (EGFR) inhibition provides downstream benefits in elastic fiber assembly and deposition in aneurysmal smooth muscle cell cultures. This work can open future possibilities for development of EGFR-targeted drug-based therapies not only for vessel wall repair in AAAs but also other proteolytically compromised elastic tissues.

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes vascular disease and associates with platelet reactivity in humans. Here, using a human genomic and proteomic approach, we identify a high affinity, disease-relevant, and potentially targetable interaction between SVEP1 and the orphan receptor Platelet and Endothelial Aggregation Receptor 1 (PEAR1). This interaction promotes PEAR1 phosphorylation and disease associated AKT/mTOR signaling in vascular cells and platelets. Mice lacking SVEP1 have reduced platelet activation, and exogenous SVEP1 induces PEAR1-dependent activation of platelets. SVEP1 and PEAR1 causally and concordantly relate to platelet phenotypes and cardiovascular disease in humans, as determined by Mendelian Randomization. Targeting this receptor-ligand interaction may be a viable therapeutic strategy to treat or prevent cardiovascular and thrombotic disease.

Thoracic aortic aneurysm (TAA) is the major morbidity/mortality factor in Marfan syndrome (MFS), a connective tissue disease caused by mutations in fibrillin‐1. We examined the impact of fibrillin‐1 (Fbn1) deficiency on the integrity and function of the tunica intima by using the Cdh5‐Cre transgene to selectively inactivate the Fbn1 gene in endothelial cells (ECs). The resulting Fbn1Cdh5‐/‐mice had a normal life span and displayed no overt signs of arterial disease. However, ultrastructural analyses of the ascending aorta of Fbn1Cdh5‐/‐mice at P18 and P45 revealed severe fragmentation of the internal elastic lamina (IEL) with biosynthetically active sub‐endothelial cells (SMCs) breaching into the intima. Additional analyses related loss of fibrillin‐1 deposition in the IEL with increased endothelial permeability, impaired EC‐dependent vascular relaxation and dysregulated gene expression in mural cells. The first two abnormalities were found in common with MFS mice that are hypomorphic for fibrillin‐1 throughout the entire vessel (Fbn1mgR/mgR mice). As these MFS mice also displayed aortic hypercontractility and a more abnormal aortic transcriptome, we concluded that Fbn1Cdh5‐/‐mice replicate the early EC‐dependent events that eventually trigger full blown TAA pathology in MFS. Preliminary analyses of single cell RNASeq data indicate that loss of intimal fibrillin‐1 altered the profile of aortic subclusters, particularly those corresponding to immune cells and ECs.

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillinmicrofibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFβ growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and −2 into the extracellular matrix in vitro . We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFβ-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms, and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with small interfering RNA (siRNA) provides greater specificity of action and improved regenerative/antiproteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cells (EaRASMCs). A siRNA dose of 12.5 nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation versus untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25 kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40 kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease-2 versus treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms. Impact statementThe elastic matrix and elastic fibers are key components of the structural extracellular matrix of elastic tissues and are essential to their stretch and recoil and to maintain healthy cell phenotype. Regeneration and repair of elastic matrix is naturally poor and impaired and is an unresolved challenge in tissue engineering. In this work, we investigate a novel gene silencing approach based on inhibiting the JNK2 gene, which provides significant downstream benefits to elastic fiber assembly and maturation. Combined with novel delivery strategies such as nanoparticles, we expect our approach to effect in situ elastic matrix repair in the future.

Aortic wall material properties are needed for computational models and for comparisons across developmental and disease states. There has been abundant work in comparing aortic material properties across disease states, but limited work across developmental states. We performed passive biaxial mechanical testing on newborn mouse aorta with (Eln+/+) and without (Eln-/-) elastin. Elastin provides elasticity to the aortic wall and is necessary for survival beyond birth in the mouse. Mechanically functional elastin is challenging to create in vitro and so Eln-/- aorta can be a comparison for tissue engineered arteries with limited elastin amounts. We found that a traditional arterial strain energy function provided reasonable fits to newborn mouse aorta and generally predicted lower material constants in Eln-/- compared to Eln+/+ aorta. At physiologic pressures, the circumferential stresses and moduli trended lower in Eln-/- compared to Eln+/+ aorta. Increased blood pressure in Eln-/- mice helps to alleviate the differences in stresses and moduli. Increased blood pressure also serves to partially offload stresses in the isotropic compared to the anisotropic component of the wall. The baseline material parameters can be used in computational models of growth and remodeling to improve understanding of developmental mechanobiology and tissue engineering strategies.

Hutchinson-Gilford progeria syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated prelamin A protein “progerin” that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially detrimental for vascular smooth muscle cells (VSMC). Vessel stiffening and aortic atherosclerosis in HGPS patients are accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here, we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNA-seq analysis shows induction of a profibrotic and pro-inflammatory aging–associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and pro-inflammatory signatures contribute to vascular stiffness in HGPS.

Large elastic arteries, such as the aorta, contain concentric layers of elastic laminae composed mainly of the extracellular matrix protein elastin. The structure of the elastic laminae could affect transmural mass transport and contribute to aortic disease progression. We studied the effects of a genetic mutation (LoxM292R/+, referred to as MU) in mice associated with ascending thoracic aortic aneurysm (TAA) on the mass transport and elastic laminae structure. Solute absent fluid flux and hydraulic conductance through the ascending aortic wall were not significantly different between groups, however solute present fluid flux, hydraulic conductance, solute flux, and solute permeability of 4 kDa FITC-dextran were significantly increased in the MU group, indicating that movement of small molecules into the aortic wall is facilitated in MU mice. Quantification from light microscopy images of the ascending aorta showed no significant differences in wall thickness, or inner elastic lamina fenestration size and density, but an increase in the number of elastic laminae breaks in the MU group. Ultrastructural comparisons from transmission electron micrographs suggest less dense and disorganized elastic laminae in MU aorta that may also contribute to the transport differences. Our results provide an initial investigation into the connections between mass transport and elastic laminae structure, specifically in a genetic mouse aneurysm model, which can be further used to understand TAA pathology and develop treatment strategies.

In the resistance artery endothelium, we show phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), signaling microdomains that regulate vasodilation. In silico data has implied PS may compete with PIP 2 binding on Kir2.1, a channel involved in vasodilatory signaling. We found 83.33% of Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate PS blocks PIP 2 activation of Kir2.1, and addition of exogenous PS blocks PIP 2 -mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries ( Eln fl/fl /Cdh5-Cre), PS localization in endothelium was disrupted and PIP 2 activation of Kir2.1 was significantly increased. Taken together, our data suggests PS enrichment to MEJs inhibits PIP 2 -mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and demonstrates the intracellular lipid localization within endothelium is an important determinant of vascular function.

In large conduit arteries, smooth muscle cell elastin produces elastic laminae, while in resistance arteries endothelial cell derived elastin is the major contributor to the internal elastic lamina (IEL). The IEL in resistance arteries separates smooth muscle and endothelium and contain distinct holes in the IEL (HIEL) where the two cell types communicate via a structure termed the myoendothelial junction (MEJ). The MEJ is an anatomical hallmark of resistance arteries that pierces through the IEL and facilitates vasodilation by movement of endothelial derived hyperpolarization (EDH) to smooth muscle. In contrast, large arteries dilate via nitric oxide (NO) and do not contain MEJs. To study the role of the IEL in resistance arteries in facilitating MEJ formation and EDH‐based vasodilation, we used a mouse with a Cdh5Cre to constitutively knockout elastin from endothelial cells (elastin ECKO). In en face preparations from third order mesenteric arteries, elastin ECKO mice had a 55.49% reduction in IEL surface area, and, importantly, completely lacked the distinct HIEL where MEJs could form. Regardless of this, en face claudin‐5 staining demonstrated a normal endothelial cell morphology. The lack of distinct HIEL, the canonical MEJ sites, led us to hypothesize vasodilatory mechanisms may be disrupted. To test this hypothesis, we first performed pressure myography on third order mesenteric arteries from elastin ECKO mice. Myogenic tone began to diverge from littermate controls starting at 80 mmHg, with preserved active, but not passive tone. For this reason, to test for changes in EDH between control and elastin ECKO, we used arteries pressurized to 60 mmHg and performed acetylcholine (Ach) dose‐response curves. We found elastin ECKO mice have decreased responses to Ach. Additionally, in the presence of 30 uM L‐NAME, the majority of dilation in elastin ECKO arteries was inhibited, compared to no inhibition with L‐NAME in the littermate controls. This indicated that NO‐mediated dilation was present in elastin ECKO mice and not littermate controls. En face immunohistochemistry demonstrated disruption of proteins localized to MEJs that regulate EDH in resistance arteries: both phosphatidylserine (See Abstract ID:R5515) and alpha globin, which is used to chelate NO. These results indicated that the disruption in the IEL, and thus canonical MEJ sites, affected the polarization of EDH‐associated proteins in the endothelium, supporting the observation of increased NO‐based signaling in elastin ECKO arteries. Overall, these data imply endothelial‐derived elastin is necessary for the functional formation of MEJs, the vasodilatory capacity of the endothelium, and the pathway of vasodilation in resistance arteries.