Endothelium

Endothelial Phenotype

Molecular mechanisms that control the induction of arterial and venous endothelial identity include upstream and downstream effectors of Wnt, Sox, and Notch pathways,¹ but little is known on the determinants that maintain this specific identity of the endothelium. van Geemen et al² bring convincing evidence that in vivo endothelial phenotype of human arteries and veins is determined by F-actin-anchored focal adhesion and biomechanical properties of extracellular matrix. In adult arteries, prominent F-actin fibers follow the orientation of fibronectin and anchor to focal adhesions recruiting integrin binding to paxillin and focal adhesion kinase, whereas in venous endothelium, F-actin fibers mostly localize cortically at cell boundaries.

Endothelial Epigenetics

Epigenetic modifications in cardiovascular diseases have raised considerable interest as so far, genome wide association studies account for only 10% of coronary artery disease inheritability.^3,4 These alterations in chromatin, which do not result from changes in DNA sequence, involve DNA methylation, RNA-based mechanisms and histone variants, and posttranslational modification, such as acetylation, methylation, or phosphorylation. Epigenetic modifications taking place in endothelial cells have been elegantly reviewed in a recent mini-series.^5–7 Current epigenetic studies focus on protein coding-genes, and it remains unknown if such modifications also regulate nonprotein-coding genes and whether or not long noncoding RNAs play a role.

Better understanding of endothelial epigenetic modifications, in particular under different flow conditions, will certainly improve the identification of new therapeutic targets in cardiovascular disease. Two studies recently summarized how disturbed proatherogenic flow upregulates DNA methyltransferases in vivo and in vitro, resulting in alterations of genome-wide DNA methylation and changes in gene expression.^6,7 For instance, the promoters of the mechanosensitive genes HoxA5, Klf3, and Klf4 are hypermethylated in disturbed flow conditions, highlighting how flow controls epigenomic methylation patterns, in turn, regulating atherosclerosis development. Shear-stress dependent regulation of endothelial noncoding RNA, in particular microRNA, as well as their transfer to neighboring cells after the release of endothelial extracellular vesicles, was recently summarized in the context of atherosclerosis.^8,9 Histone posttranslational modification by acetylation results from the balance between acetylase and deacetylase enzyme activities and involves the large family of histone deacetylases (HDAC), which includes sirtuins¹⁰ and HDAC. Further studies will determine how proatherogenic and atheroprotective flow conditions regulate endothelial HDAC and NAD-dependent HDAC sirtuin-1 that protects against oxidative stress⁷ and multiple other parameters.

Deciphering endothelial epigenetic modifications will also help biologists understand more fundamental observations such as molecular basis of endothelial cell-specific gene expression.⁵ Okada et al¹¹ have tested this hypothesis to decipher the specific endothelial expression of Roundabout (Robo4), a transmembrane receptor involved in migration, proliferation, angiogenesis, and stabilization of the vasculature. The ubiquitous expression of Robo4 transcription factors GABP and S1P, which regulate its promoter activation, cannot explain Robo4 specific endothelial localization. Okada et al¹¹ demonstrate that the endothelial specific DNA methylation pattern of Robo4 proximal promoter during cell differentiation regulates Robo4 endothelial specific expression. This finding might be important as other endothelial specific genes, including von Willebrand Factor (vWF), E-selectin, VEGF receptors 1 and 2, and platelet endothelial cell adhesion molecule are also regulated by non–cell-type specific transcription factors.

Regulation of Vascular Tone

Because the seminal work from Furchgott and Zawadzki¹² demonstrating the obligatory role of the endothelial cell in the control of vascular tone, the contribution of different mediators, including nitric oxide (NO), to endothelium-dependent responses has been extensively investigated.^13,14 Schuler et al¹⁵ have established a noninvasive method in mice to assess flow- and NO-dependent vasodilation of murine femoral arteries. This approach might be extremely useful to investigate in vivo endothelial function in murine models of vascular diseases and in genetically modified mice. In humans, endothelium-dependent regulation of vascular tone seems to be affected by ethnic origin. In particular, Ozkor et al¹⁶ report ethnic differences in the availability of NO and endothelium-derived hyperpolarizing factor. They show that the contribution of basal and stimulated release of NO to regulation of vascular tone is greater in white than in healthy black individuals. In addition, endothelium-derived hyperpolarizing factor partially compensates for the reduced NO contribution in black subjects. Vascular tone is also regulated by adipokines and yet unidentified factors that are released from perivascular adipose tissue. These relaxing factors stimulate potassium channel opening in vascular smooth muscle cells and could help fight vascular dysfunction in obesity and hypertension.¹⁷ Melanocortin-1 receptor (MRC-1), which regulates melanin pigmentation in the skin, has been also identified in endothelial cells where it increases NO availability. Rinne et al¹⁸ report that alterations in MRC-1 signaling in mice and in humans leads to impaired endothelial-dependent vasodilation and NO availability and to increased arterial stiffness. These findings highlight the role of MRC-1 in the regulation of vascular tone.

Increasing high-density lipoprotein levels after inhibition of cholesteryl ester transfer protein activity improves endothelial repair and function in a model of balloon vascular injury.¹⁹ In normocholesterolemic rabbits, cholesteryl ester transfer protein inhibition reduced intimal thickening and regenerates a functional endothelium through mechanisms involving scavenger receptor B1 and phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt. Whether or not this beneficial effect also occurs in humans following treatment with cholesteryl ester transfer protein inhibitors remains an open question.

Using mice specifically deficient in endothelial AMP-activated protein kinase (AMPK) isoform α-1 or -2, Enkhjargal et al²⁰ demonstrate that AMPK-α-1, the major AMPK isoform expressed in endothelial cells, mediates endothelium-dependent hyperpolarizations in resistance arteries, and regulates blood pressure and coronary flow in vivo. Presence of MRC-1 in endothelial cells increases NO availability.¹⁸ This receptor is abundantly expressed in the skin where it regulates melanin pigmentation. Alterations in MRC-1 signaling impair endothelial and NO-dependent vasodilation in mice and in humans, leading to increased arterial stiffness.¹⁸ These findings highlight the role of endothelial MRC-1 in the regulation of vascular tone. Expression of endothelial nitric oxide synthase (eNOS) is regulated by several exogenous stimuli, including tumor necrosis factor-a, which destabilizes eNOS mRNA leading to decreased eNOS expression. Tumor necrosis factor-a increases the expression of the cysotolic protein polypyrimidine tract-binding protein 1, which specifically binds to the eNOS 3′ untranslated region. This results in decreased eNOS expression and impaired endothelial vasodilatory responses.²¹ Therefore, modulation of tract-binding protein 1 expression might be of interest in treating endothelial dysfunction. Angiotensin II stimulates the endothelial expression and occupancy of histone H3K4 trimethyltransferase SET1 on the endothelin-1 promoter, activating the transcription of this potent vasoconstrictor.²² Specific-endothelial deficiency in SET1 prevents angiotensin II-induced release of endothelin-1 and abrogated hypertrophy of cultured cardiomyocytes. These findings reinforce the interest for SET1 inhibitors for treatment of cardiomyopathy.

Angiogenesis, Arteriogenesis

Efficient endothelial repair after endothelial damage or injury is a critical step to prevent adverse arterial remodeling, thrombosis, and atherosclerosis.^23,24 In this regard, microRNA seem as an interesting tool for improving angiogenesis.²⁴ Cao et al²⁵ investigated the therapeutic angiogenic effect of miR126-3p using ultrasound targeted microbubble destruction, a noninvasive technique for targeted vascular transfection of plasmid DNA, including microRNAs. In their study, transfection of miR-126-3p before and after rat left femoral artery ligation improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels with no effect on miR-126-5p. Altogether, these findings support the potential for ultrasound targeted microbubble destruction for microRNA delivery, in particular for therapeutic angiogenesis. Manipulating insulin-like growth factor-1 receptor expression in bone marrow cells seems also of interest to improve endothelial repair after arterial wall injury.²⁶ Insulin-like growth factor-1 receptor haploinsufficiency in bone marrow cells accelerates endothelial regeneration in a murine model of vascular wire injury and does not alter the formation of atherosclerotic lesions. Despite their lower number, angiogenic progenitors display enhanced adhesion, increased release of insulin-like growth factor-1 and enhanced angiogenic capacity.

The platelet glycoprotein Iba (GPIba) has been identified as a critical mediator of transient platelet adhesion to endothelial cells and of leukocyte accumulation in the perivascular space of collateral vessels.²⁷ These findings underline the role of GPIba in arterial remodeling and arteriogenesis. In addition, inhibition or loss of function of GPIba jeopardizes reperfusion recovery. Using a murine model of arteriolar ligation, Bruce et al²⁸ demonstrate the unexpected and rapid entry of circulating CX3CR1⁺ monocytes at the postcapillary venous site to promote arteriogenesis.

Recent findings indicate that the Jagged1/Notch signaling regulates not only developmental angiogenesis but also plays an active role in adult angiogenesis.²⁹ By blocking Dll4 signaling through Notch1, Jagged1 allows endothelial cell growth by activating VEGF. In addition, Jagged1 binds to Notch4 and triggers maturation of the newly formed vessels. Furthermore, VEGF receptor internalization and signaling contribute to the angiogenic growth of blood vessels. Full VEGF receptor signaling and recycling of VEGFR2 to the endothelial cell surface requires the clathrin-associated sorting proteins numb and numb-like.³⁰

Finally, Pi et al³¹ bring strong evidence that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are novel-positive regulators of endothelial migration and angiogenesis induced by stromal cell–derived factor 1α a potent angiogenic chemokine. Reactive oxygen species generated by NADPH oxidases oxidize and inhibit the protein tyrosine phosphatases MKP7, thereby regulating stromal cell–derived factor 1α–dependent JNK3 activity and angiogenesis. Therefore, intracellular redox balance is critical for stromal cell–derived factor 1α–induced endothelial migration and angiogenesis.

Hypoxia/Reoxygenation

Although mitochondrial content in endothelial cells is rather modest when compared with other cell types, recent investigations suggest that mitochondrial dynamics affect endothelial cell function.³² Both studies from Haslip et al³³ and He et al³⁴ bring new evidence regarding the impact of endothelial mitochondrial function in vascular pathologies associated with hypoxia.

First, the interaction between mitochondria and the endoplasmic reticulum plays an important role in endothelial cells during reperfusion-injury.³⁴ Mitochondria serve as calcium buffer sites and regulate calcium uptake and release by the endoplasmic reticulum. During cell stimulation, continuous flux of calcium through mitochondria is needed for store-operated entry and endoplasmic reticulum calcium store refilling. Acetylcholine, a neurotransmitter released during vagal nerve stimulation, decreases both intracellular and mitochondrial Ca²⁺ overload, and protects endothelial cells from hypoxia/reperfusion injury.³⁴ This effect likely results from disruption of the endoplasmic reticulum/mitochondria crosstalk by limiting the interaction between mitofusin-2 and the complex voltage-dependent anion channel-1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1. Second, exposure to intermittent hypoxia of mice deficient in endothelial mitochondrial uncoupling protein-2 leads to the development of pulmonary hypertension.³³ This effect results from excessive phosphatase and tensin homolog (PTEN)–induced putative kinase 1-induced mitophagy, inadequate mitochondrial biogenesis, and subsequent endothelial apoptosis. Inhibiting PTEN-induced putative kinase 1 pathway in mice deficient in endothelial uncoupling protein-2 prevents pulmonary hypertension.³³ As endothelial cells from patients with pulmonary hypertension have a similar phenotype to uncoupling protein-2-deficient endothelial cells, targeting the uncoupling protein-2-PTEN–induced putative kinase 1 pathway might be of therapeutic interest in this pathology.

Flow Sensing, Shear Stress

The regulation of endothelial function and proatherogenic phenotype by mechanical forces including shear stress was recently reviewed in ATVB.^9,35,36 Until not too long ago, the effect of shear stress on endothelial cells had focused on changes in the steady-state levels of mRNAs. However, several new findings point out that epigenetic mechanisms of transcription, changes in mRNA stability and RNA translational efficiency, mediated by altered microRNA expression are other important mechanisms regulating endothelial gene function in different hemodynamic conditions.³⁷ Furthermore, Murphy and Hynes³⁸ provide new evidence that disturbed blood flow affects mRNA splicing of a functionally important endothelial cell gene product. In their study, they demonstrate that disturbed flow leads to changes in the mRNA splicing of endothelial fibronectin and that these alternatively spliced exons protect the vasculature from inflammation caused by disturbed flow.^38,39 Shear stress also affects endothelial metabolism. Exposure of endothelial cells to laminar flow impairs their capacity to uptake glucose and their mitochondrial content in a Kruppel-like factor (KLF)-2 dependent manner.⁴⁰ This effect results from the inhibition caused by shear stress of key glycolytic enzymes, and in particular, repression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) promoter. All together these findings demonstrate that shear stress repression of endothelial metabolism is mediated by KLF2 and that PFKFB3 seems as an underestimated regulator of endothelial cell phenotype. These findings might help explain the switch of endothelial cell activation and angiogenesis back to a quiescent endothelium once blood flow is established.

Endothelial senescence has been observed in atheroprone areas of the vasculature. Warboys et al⁴¹ provide now a mechanistic link between endothelial shear stress and cell senescence by demonstrating that disturbed or low flow promotes endothelial senescence via the p53–p21 pathway. As this effect can be inhibited by sirtuin-1, pharmacological activation of sirtuin1 may promote endothelial health by suppressing endothelial senescence in atheroprone areas.⁴¹ Flow-dependent outward remodeling of resistance arteries is a key adaptive process declining with age. Tarhouni et al⁴² demonstrate that estrogen deprivation rather than age impairs flow mediated remodeling and that timing for estrogen replacement is important to improve this response.

Endothelial Inflammation

Leukocyte recruitment at inflammatory sites is important for an appropriate inflammatory response in cardiovascular pathologies. Zuchtriegel et al⁴³ identified specific-spatiotemportal expression patterns of selectins in inflammatory cells and endothelial cells. For instance, P-selectin, L-selectin, and P-selectin-glycoprotein ligand regulate neutrophil and monocyte flux, whereas endothelial E-selectin controls the rolling velocity of inflammatory monocytes. These specific-expression patterns of selectins associate with the sequential infiltration of inflammatory leucocytes and collectively enable the sequential extravation of leucocytes to the inflammed tissue. As endothelial peroxisome proliferator-activated receptor gamma receptor activation downregulates P-selectin expression and impairs leukocyte-endothelial interaction, this pathway might be an interesting therapeutic target to protect against thrombosis.⁴⁴ On the contrary, endothelial angiopoietin-like-protein-2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.⁴⁵ Psoriasis is an inflammatory skin disease associated with increased cardiovascular mortality. A mouse psoriasis model where interleukin-17A (IL-17A) was overexpressed in the dermis was used to test the hypothesis that increased IL-17A production in the skin may systemically cause vascular dysfunction. This model overexpressing dermal IL-17A was characterized by infiltration of myeloperoxidase⁺CD11b⁺GR1⁺F4/80⁻ inflammatory cells, increased oxidative stress, systemic endothelial dysfunction, and hypertension.⁴⁶ The deleterious consequences of IL-17A overexpression were improved by neutralizing cytokines downstream of IL-17A such as tumor necrosis factor-a and IL-6, or depleting GR1⁺ inflammatory cells. These finding are in favor of the causal role of IL-17A–dependent dermal inflammation in systemic vascular alterations observed in psoriasis.

Endothelial inflammation is associated with increased permeability. Near-infrared flurorescence imaging seems as an interesting new tool to monitor endothelial permeability in large vessel, as it leaves tissues intact for subsequent histological analysis of quantification of leucocytes subpopulations by flow cytometry.⁴⁷ A nanoparticle-based imaging strategy was also developed to identify pathological endothelial cells in a murine model of arteriovenous fistula.⁴⁸ In this model, nanoparticles labeled preferentially sites expressing high levels of VCAM-1 and characterized by increased permeability. Therefore, these nanoparticles identify areas with a pathological endothelium where neointimal hyperplasia subsequently develops in arteriovenous fistula. This strategy could be of potential interest to monitor therapeutic interventions to decrease the failure of arteriovenous fistula resulting from in flow stenosis in patients with end-stage renal disease.

Inflammatory conditions can form large vWF fibers, which immobilize on endothelial surface to form highly adhesive subtrate under shear conditions. Grässle et al elegantly show that vWF directly binds and immobilizes extracellular DNA released from leucocytes (neutrophil extracellular traps). DNA-bound vWF decreases platelet binding to vWF and might be a linker for leukocyte adhesion to endothelial cells, favoring leukocyte extravasation, and inflammation.⁴⁹ Cocaine consumption leads to endothelial dysfunction and accelerated atherosclerosis. Endothelial cells exposed to cocaine or plasma from chronic cocaine consumers were more proadhesive, with increased von Willebrand deposition, enhancing platelet binding, an effect prevented by statin treatment.⁵⁰ Atherosclerotic stimuli increase Arginase-2 expression, leading to eNOS uncoupling. The specific HDAC2 is a critical regulator of endothelial Arginase2 transcription. HDAC2 limits Arginase2 transcription in healthy conditions, but this control is impaired in proatherogenic conditions and oxidative injury. Therefore, HDAC2 activation or overexpression could represent a new therapy for endothelial dysfunction in atherosclerosis.⁵¹ Finally, deficiency in scavenger receptor B1 in Ldlr^−/− mice is associated with increased endothelial VCAM and ICAM expression in coronary arteries and augmented levels of circulating inflammatory markers, including increased proportions of Ly6C(hi) and Ly6C(int) monocytes.⁵²

Endothelium in Diseases