scholarly journals Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Darian Williams ◽  
Marwa Mahmoud ◽  
Renfa Liu ◽  
Aitor Andueza ◽  
Sandeep Kumar ◽  
...  

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

2021 ◽  
Author(s):  
Darian Williams ◽  
Marwa Mahmoud ◽  
Renfa Liu ◽  
Aitor Andueza ◽  
Sandeep Kumar ◽  
...  

Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (dflow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Methods and Results: Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10- expressing plasmids inhibited atherosclerosis in ApoE-/- mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10's anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. Conclusion: KLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.


Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Darian Williams ◽  
Marwa Mahmoud ◽  
Hanjoong Jo

Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow ( d-flow ) while the straight regions exposed to stable flow ( s-flow ) are protected. The proatherogenic and atheroprotective effects of flow are mediated in large part by the global changes in endothelial cell gene expression, which regulate endothelial dysfunction and inflammation. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10) as one of the most flow-sensitive genes in arterial endothelial cells using the partial carotid ligation model of d-flow -induced atherosclerosis. KLK10 is a secreted serine protease, but its role in endothelial function and atherosclerosis is unknown. Methods/Results: Here, we validated that KLK10 was upregulated under s-flow conditions and downregulated under d-flow conditions using the in vivo mouse models and in vitro studies using endothelial cells (ECs). Through in vitro functional studies using ECs, we found that KLK10 produced by s-flow protected against endothelial inflammation and permeability dysfunction. Furthermore, treatment with rKLK10 or overexpression of KLK10 plasmids in vivo decreased endothelial inflammation the mouse model. Further, rKLK10 injection or ultrasound-mediated transfection of KLK10 plasmids in the hind leg muscles led to inhibition of atherosclerosis in ApoE-/- mice with the partial carotid ligation surgery. Studies using the pharmacological inhibitors and siRNAs showed that the anti-inflammatory effects of KLK10 was mediated by the Protease Activated Receptors 1 and 2, but without directly cleaving them. Further studies show that KLK10’s anti-inflammatory effect was mediated by the NFκB and VCAM1 and ICAM1 expression pathway. In addition, immunostaining showed that KLK10 expression is significantly reduced in human coronary arterial sections with atherosclerotic plaques compared to the non-diseased controls. Conclusions: We found that KLK10 is a potent flow-sensitive secreted protein, which serve as a novel anti-inflammatory and anti-atherogenic factor. KLK10 may be a potential anti-atherogenic therapeutic.


2018 ◽  
Vol 24 (3) ◽  
pp. 278-287 ◽  
Author(s):  
Shengnan Zhao ◽  
Minglu Liang ◽  
Yilong Wang ◽  
Ji Hu ◽  
Yi Zhong ◽  
...  

The vascular endothelium is a continuous layer of flat polygonal cells that are in direct contact with the blood and participate in responses to inflammation. Chrysin is a flavonoid compound extracted from plants of the genus Asteraceae with a wide range of pharmacological activities and physiological activities. Here, we studied the effects of chrysin on the regulation of the proadhesion and pro-inflammatory phenotypes of the endothelium both in vitro and in vivo. Our results revealed that chrysin strongly inhibited Tohoku Hospital Pediatrics-1 (THP-1) cell adhesion to primary human umbilical vein endothelial cells and concentration-dependently attenuated interleukin 1β-induced increases in intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin messenger RNA levels and ICAM-1 and VCAM-1 protein levels. Previous studies reported that nuclear factor κB (NF-κB) is important in the inflammatory response in endothelial cells, particularly in regulating adhesion molecules, and our data shed light on the mechanisms whereby chrysin suppressed endothelial inflammation via the NF-κB signaling pathway. In addition, our in vivo findings demonstrated the effects of chrysin in the permeability and inflammatory responses of the endothelium to inflammatory injury. Taken together, we conclude that chrysin inhibits endothelial inflammation both in vitro and in vivo, which could be mainly due to its inhibition of NF-κB signaling activation. In conclusion, chrysin may serve as a promising therapeutic candidate for inflammatory vascular diseases.


2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Helong Zhao ◽  
Appakkudal Anand ◽  
Ramesh Ganju

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.


2021 ◽  
Author(s):  
Qiuhua Yang ◽  
Jiean Xu ◽  
Qian Ma ◽  
Zhiping Liu ◽  
Yaqi Zhou ◽  
...  

Overnutrition-induced endothelial inflammation plays a crucial role in high fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficient mice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.


Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 439
Author(s):  
Katrine T. Callesen ◽  
Alma Yuste-Montalvo ◽  
Lars K. Poulsen ◽  
Bettina M. Jensen ◽  
Vanesa Esteban

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.


Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2210
Author(s):  
Maria Pompea Antonia Baldassarre ◽  
Pamela Di Tomo ◽  
Giorgia Centorame ◽  
Assunta Pandolfi ◽  
Natalia Di Pietro ◽  
...  

Myo-inositol (Myo) improves insulin resistance, glucose metabolism, and helps gestational diabetes (GDM) management. GDM is associated with a pro-inflammatory state and increased oxidative stress, which are both involved in vascular damage in diabetes. Our aim was to study Myo anti-inflammatory/antioxidant potential effects on an in vitro model of human umbilical vein endothelial cells (HUVECs). To this end, monocyte cell adhesion to HUVECs, adhesion molecule membrane exposure, and oxidative stress levels were determined in cells from control (C-) and GDM women treated during pregnancy either with diet only (GD-) or with diet plus Myo (GD+Myo). To deeply study the vascular effects of Myo, the same evaluations were performed in C- and GD-HUVECs following 48 h in vitro stimulation with Myo. Notably, we first observed that GD-HUVECs obtained from women assuming Myo supplementation exhibited a significantly decreased number of monocytes that adhered to endothelial cells, less adhesion molecule exposure, and lower intracellular reactive oxygen species (ROS) levels in the basal state as compared to GD-HUVECs obtained from women treated by diet only. This Myo anti-inflammatory/antioxidant effect was confirmed by 48 h in vitro stimulation of GD-HUVECs as compared to controls. Altogether, these results strongly suggest that Myo may exert protective actions against chronic inflammation induced by endothelial dysfunction in diabetes.


Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3498-3506 ◽  
Author(s):  
Graeme M. Birdsey ◽  
Nicola H. Dryden ◽  
Valerie Amsellem ◽  
Frank Gebhardt ◽  
Kapil Sahnan ◽  
...  

Abstract Tight regulation of the balance between apoptosis and survival is essential in angiogenesis. The ETS transcription factor Erg is required for endothelial tube formation in vitro. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVECs), using antisense oligonucleotides, resulted in detachment of cell-cell contacts and increased cell death. Inhibition of Erg expression by antisense in HUVECs also lowered expression of the adhesion molecule vascular endothelial (VE)–cadherin, a key regulator of endothelial intercellular junctions and survival. Using chromatin immunoprecipitation, we showed that Erg binds to the VE-cadherin promoter. Furthermore, Erg was found to enhance VE-cadherin promoter activity in a transactivation assay. Apoptosis induced by inhibition of Erg was partly rescued by overexpression of VE-cadherin–GFP, suggesting that VE-cadherin is involved in the Erg-dependent survival signals. To show the role of Erg in angiogenesis in vivo, we used siRNA against Erg in a Matrigel plug model. Erg inhibition resulted in a significant decrease in vascularization, with increase in caspase-positive endothelial cells (ECs). These results identify a new pathway regulating angiogenesis and endothelial survival, via the transcription factor Erg and the adhesion molecule VE-cadherin.


1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.


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