Vascular Endothelial Cell Interactions Research Articles

Interaction of vascular endothelial cells with hydrophilic fullerene nanoarchitectured structures in 2D and 3D environments

ABSTRACT The interaction between diverse nanoarchitectured fullerenes and cells is crucial for biomedical applications. Here, we detailed the preparation of hydrophilic self-assembled fullerenes by the liquid-liquid interfacial precipitation (LLIP) method and hydrophilic coating of the materials as a possible vascularization strategy. The interactions of vascular endothelial cells (ECs) with hydrophilic fullerene nanotubes (FNT-P) and hydrophilic fullerene nanowhiskers (FNW-P) were investigated. The average length and diameter of FNT-P were 16 ± 2 μm and 3.4 ± 0.4 μm (i.e. aspect ratios of 4.6), respectively. The average length and diameter of FNW-P were 65 ± 8 μm and 1.2 ± 0.2 μm (i.e. aspect ratios of 53.9), respectively. For two-dimensional (2D) culture after 7 days, the ECs remained viable and proliferated up to ~ 420% and ~ 400% with FNT-P and FNW-P of 50 μg/mL, respectively. Furthermore, an optimized chitosan-based self-healing hydrogel with a modulus of ~400 Pa was developed and used to incorporate self-assembled fullerenes as in vitro three-dimensional (3D) platforms to investigate the impact of FNT-P and FNW-P on ECs within a 3D environment. The addition of FNW-P or FNT-P (50 μg/mL) in the hydrogel system led to proliferation rates of ECs up to ~323% and ~280%, respectively, after 7 days of culture. The ECs in FNW-P hydrogel displayed an elongated shape with aligned morphology, while those in FNT-P hydrogel exhibited a rounded and clustered distribution. Vascular-related gene expressions of ECs were significantly upregulated through interactions with these fullerenes. Thus, the combined use of different nanoarchitectured self-assembled fullerenes and self-healing hydrogels may offer environmental cues influencing EC development in a 3D biomimetic microenvironment, holding promise for advancing vascularization strategy in tissue engineering.

The effects of the interaction of vascular endothelial cells and vascular smooth muscle cells on PCSK9 and NF-kB protein expression in vascular smooth muscle cells

Amaç: Ateroskleroz dünyada görülen en sık ölüm sebebidir. Ateroskleroz inflamatuar bir hastalık olup, damar duvarı hücrelerinde belirgin şekil ve fonksiyon değişikliklerine neden olur. Damar duvarındaki ana hücreler olan endotel hücreleri ile düz kas hücrelerinin ateroskleroz sırasında iletişim halinde oldukları bilinen bir gerçek olmakla beraber, bu iletişimin mekanizmaları net değildir. PCSK9 ise hiperlipidemi etyolojisinde yer alan bir molekül olmakla birlikte, damar duvar hücrelerinde ateroksleroz sırasında artar ve damar duvar hücrelerine direk aterosklerotik etkileri vardır. Çalışmamızda aterojenik etkenlere maruz kalan endotel hücrelerinin, düz kas hücrelerinde yaptığı değişiklikleri ve inflamasyonun en önemli molekülü olan NF-kB ve direct aterosklerotik etkileri son zamanlarda keşfedilmiş olan PCSK9 protein ekspresyonlarına olan etkilerini araştırdık. Metod: İnsan umblikal veninden izole edilen hücreler (HUVEC) ayrı ayrı 24 saat boyunca 300 µM hidrojen peroksit (H2O2), 40 µg/ml okside LDL (Ox-LDL) %5 sigara duman ekstraktı (SDE) ile inkübe edildi, 24 saat sonra aterojenik etkenlere maruz kalan endotel hücrelerinin süpernatantları uygun işlemlerden sonra insan aortic düz kas hücrelerinin (HAVSMC) inkübe edilmesi için kullanıldı. Düz kas hücrelerinden 48 saat sonra protein ekstraksiyonu yapılarak Western Blot yöntemiyle analiz edildi. Bulgular: Aterojenik maddelere maruz kalan HUVEC’lerde canlılığın korunduğu saptandı. Bu maddelere maruz kalan endotel hücre süpernatantları ile inkübe edilen HASMC’de belirgin bir morfolojik değişiklik saptandı. %5 SDE ve 300 µM H2O2’ye maruz kalan endotel hücre süpernatantları ile inkübe edilen HASMC’lerde NF-kB (sırasıyla p=0.024,p=0.043)ve PCSK9 protein (p=0.034, p=0.0137) ekspresyonları artmış bulundu. Ox-LDL’ye maruz kalan endotel hücre süpernatantı ile inkübe edilen HASMC’lerde ise PCSK9 ekspresyonu artmış olup (p=0.037), NF-kB ekspresyonunda anlamlı bir artış izlenmemiştir. (p=0.89) Sonuç: Çalışmamızın sonucunda aterojenik etkenlere maruz kalan endotel hücrelerinin, damar düz kas üzerinde değişikliklere neden olduğu,damar düz kas hücrelerindeki PCSK9 ve NF-kB ekspresyonunu arttırdığı gözlenmiştir.

Ninjurin1 Deletion in NG2-Positive Pericytes Prevents Microvessel Maturation and Delays Wound Healing

The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.

PS 01-25] NINJURIN1 IS A NOVEL FACTOR TO MEDIATE VESSEL MATURATION THROUGH ENDOTHELIAL-PERICYTES INTERACTIONS

Objective : It is known that abnormality of adventitial microvessel (vasa vasorum) is involved in vascular remodeling and progression of atherosclerosis. Although the normalization/maturation of micorvessels is attractive approach for anti-atherosclerosis, the knowledge about the mechanisms for microvascular normalization is limited. Interaction of vascular endothelial cells (ECs) and pericytes (PCs) is crucial to vascular maturation. Recently, we have reported that Ninjrin1(Ninj1) in PCs inhibit ECs growth. However, the role of Ninj1 in patho-physiological angiogenesis is still unclear . Accordingly, we examined the effects of Ninj1on the formation of neovessels using mouse hind limb ischemia (HLI) model. Design and Method: Capillary ECs and PCs were prepared from adventitia of mouse femoral artery . Cell cluster or 3D-gel assay was performed by mixture of ECs and PCs in non-adherent or Matri-gel-coated wells. Knock down of Ninj1 in cells or tissues were induced by transfection of Ninj1-SiRNA or biodegradable microspheres releasing Ninj1-siRNA. Blood flow recovery and formation of microvessels in limbs were measured by Doppler flow meter and staining with anti-CD31 or lectin-FITC. Results: Ninj1 was expressed in microvessels, both dominantly PCs and ECs. Cell cluster assay demonstrated that down-regulation of Ninj1 in PCs or ECs reduced the PC-EC interaction. When ECs were co-incubated with PCs in 3D-gel, they made matured mirovessel structure, i.e. EC-tube wrapped with PCs. Down-regulation of Ninj1 in PCs or ECs reduced inhibited the formation of this matured vessel structure. Ninj1 was expressed in microvessels within skeletal muscle tissues, and their expression was enhanced after HLI operation. When the expression of Ninj1 was inhibited by intra-muscular injection of Ninj1-siRNA leads the formation of lectin/CD31-positive functional matured microvessels and the blood flow recovery was decreased. Conclusions: Ninj1 is important for the association between PCs and ECs to form functional matured vessels. This finding would provide insights into the therapy for atherosclerosis in addition to ischemic diseases.

Biphasic modulation of paracellular claudin-5 expression in mouse brain endothelial cells is mediated through the phosphoinositide-3-kinase/AKT pathway.

Blood-brain barrier (BBB) integrity is compromised in many central nervous system disorders. Complex astrocyte and vascular endothelial cell interactions that regulate BBB integrity may be disturbed in these disorders. We previously showed that systemic administration of 3-chloropropanediol [(S)-(+)-3-chloro-1,2-propanediol] induces a transitory glial fibrillary acidic protein-astrocyte loss, reversible loss of tight junction complexes, and BBB integrity disruption. However, the intracellular signaling mechanisms that induce BBB integrity marker loss are unclear. We hypothesize that 3-chloropropanediol-induced modulation of tight junction protein expression is mediated through the phosphoinositide-3-kinase (PI3K)/AKT pathway. To test this hypothesis, we used a mouse brain endothelial cell line (bEnd.3) exposed to 3-chloropropanediol for up to 3 days. Results showed early reversible loss of sharp paracellular claudin-5 expression 90, 105, and 120 minutes after 3-chloropropanediol (500 μM) treatment. Sharp paracellular claudin-5 profiles were later restored, but lost again by 2 and 3 days after 3-chloropropanediol treatment. Western blot and immunofluorescence studies showed increased p85-PI3K expression and transitory increased AKT (Thr308) phosphorylation at 15 and 30 minutes after 3-chloropropanediol administration. PI3K inhibitors LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; 2.5-25 μM] and PI-828 [2-(4-morpholinyl)-8-(4-aminopheny)l-4H-1-benzopyran-4-one; 0.1-10 μM] prevented the 3-chloropropanediol-induced AKT (Thr308) phosphorylation and both early and late loss of paracellular claudin-5. However, AKT inhibitors only prevented the early changes in claudin-5 expression. This mechanistic study provides a greater understanding of the intracellular signaling pathways mediating tight junction protein expression and supports a hypothesis that two independent pathways triggered by PI3K mediate early and late loss of paracellular claudin-5 expression.

Research of the degradation products of chitosan's angiogenic function

Angiogenesis is of great importance in tissue engineering and has gained large attention in the past decade. But how it will be influenced by the biodegradable materials, especially their degradation products, remains unknown. Chitosan (CS) is a kind of naturally occurred polysaccharide which can be degraded in physiological environment. In order to gain some knowledge of the influences of CS degradation products on angiogenesis, the interaction of vascular endothelial cells with the degradation products was investigated in the present study. The CS degradation products were prepared by keeping CS sample in physiological saline aseptically at 37 °C for 120 days. Endothelial cells were co-cultured with the degradation products and the angiogenic cell behaviors, including cell proliferation, migration and tube-like structure (TLS) formation, were tested by MTT assay, cell migration quantification method (CMQM), and tube-like structure quantification method (TLSQM) respectively. Furthermore, mRNA expressions of vascular endothelial growth factor (VEGF) and matrix metallo proteinase (MMP-2) were determined by real-time reverse transcriptional polymerase chain reaction (RT-PCR). Physiological saline served as a negative control. As the results showed, the degradation products obtained from 20th to 60th day significantly inhibited the proliferation, migration, and TLS formation of endothelial cells. However, degradation products of the first 14 days and the last 30 days were found to be proangiogenic. At the molecular level, the initial results indicated that the mRNA expressions of VEGF and MMP-2 were increased by the degradation products of 7th day, but were decreased by the ones of 60th day. According to all the results, it could be concluded that the angiogenic behaviors of endothelial cells at both cellular and molecular level could be significantly stimulated or suppressed by the degradation products of CS and the influences are quite time-dependent.

Regulation of secretion and clearance of C-type natriuretic peptide in the interaction of vascular endothelial cells and smooth muscle cells

To clarify the significance of C-type natriuretic peptide in the interaction between endothelial cells and vascular smooth muscle cells by investigating the endothelial production of C-type natriuretic peptide and the clearance mechanism of C-type natriuretic peptide using the endothelial cells-smooth muscle cells co-culture system. Secretion of C-type natriuretic peptide in the direct co-culture of endothelial cells with smooth muscle cells elicited as much as a 60-fold increase compared with endothelial cells alone. The accumulation of intracellular cyclic GMP in the co-culture was consequently increased and the elevation of cyclic GMP level in the co-culture was abolished by the anti-C-type natriuretic peptide monoclonal antibody. The elevated cyclic GMP production in the co-culture was abolished by the anti-transforming growth factor-beta neutralizing antibody. Candoxatrilat (10(-6)-10(-4) mol/l), a neutral endopeptidase inhibitor, dose-dependently increased the concentrations of C-type natriuretic peptide in the culture medium with endothelial cells alone, but not in the endothelial cells-smooth muscle cells co-culture. The transcript of neutral endopeptidase messenger RNA was detected in endothelial cells but not in smooth muscle cells by reverse transcriptase polymerase chain reaction. Treatment with C-atrial natriuretic factor4-23 (10(-9)-10(-6) mol/l), the specific ligand for the clearance receptor of the natriuretic peptides, resulted in dose-dependent augmentation of C-type natriuretic peptide concentration and concomitant intracellular cyclic GMP production in the endothelial cells-smooth muscle cells co-culture but not in endothelial cells alone. The present study demonstrated that direct interaction between endothelial cells and smooth muscle cells augments C-type natriuretic peptide secretion from endothelial cells through transforming growth factor-beta activation, and revealed that the enzymatic degradation is responsible for the steady state level of C-type natriuretic peptide in endothelial cells alone and that the receptor-mediated clearance mainly determines the augmented level of C-type natriuretic peptide in the interaction between endothelial cells and smooth muscle cells. The results taken together raise the possibility that endothelial C-type natriuretic peptide might play a role in regulation of vascular tone and remodelling.

The interaction of vascular endothelial cells and dorsal root ganglion neurites is mediated by vitronectin and heparan sulfate proteoglycans

The interaction of peripheral nerve and blood vessels during development was studied by using DRG explant culture plated on confluent monolayer of vascular endothelial cells (VEC). The comparison of neurite length on various substrates showed a preference of DRG neurites in the following order; thrombospondin > laminin, vitronectin > fibronectin, VEC monolayer > collagen I, rat astrocyte monolayer. On layers of fibroblasts (3T3) or gliomas (C6), neurite extention was not observed. To identify the neurite outgrowth promoting adhesion molecules on VEC surface, several antibodies and synthetic peptides were added to the culture medium of DRG. With vitronectin antibody or with peptides containing the Arg-Gly-Asp (RGD) sequence, 30–40% of neurite outgrowth was inhibited and these two effects were not additive. Therefore, a part of neurite outgrowth in this system is mediated by vitronectin in RGD dependent manner. Another molecule which promotes neurite outgrowth on VEC was identified by a new monoclonal antibody (MAb) EC1. In the Western blot analysis, the immunoreactive band which was over 400 kDa was intensified by guanidine HCl extraction. EC1 immunoreactive band disappeared after the treatment of heparitinase but not with other glycolyases, indicating that EC1 antigen is heparan sulfate proteoglycan(s). The DRG neurite outgrowth was inhibited by MAb EC1 by about 30–40%. By the combination of MAb EC1 and RGD peptide, the neurite outgrowth in explant culture was inhibited by about 50%, and in DRG dissociated culture nearly 100% inhibition was observed. Thus, for the DRG neurite elongation on VEC, vitronectin and heparan sulfate proteoglycan(s) are playing crucial roles.

Integrin overexpression induced by high glucose and by human diabetes: potential pathway to cell dysfunction in diabetic microangiopathy.

The nature of the process leading to the acellular nonperfused capillaries of diabetic microangiopathy remains unknown. Because these capillaries manifest thickened basement membranes, we asked whether the process causing deposition of excess extracellular matrix in diabetes modifies cell-matrix interactions in a direction that would compromise cell renewal. In 44 individual isolates of human umbilical vein endothelial cells we observed that high glucose concentrations (30 mM) induce coordinate increases in the levels of mRNAs encoding fibronectin and the fibronectin-specific integrin receptor alpha 5 beta 1 as well as in the cognate proteins. Expression of the integrin subunit alpha 3, component of the alpha 3 beta 1 polyspecific receptor for fibronectin, laminin, and collagen, was also up-regulated by high glucose. Overexpression of integrins correlated with increased cell attachment to exogenous fibronectin and laminin as well as to complex matrix. Moreover, cells exhibited firmer steady-state adhesion to their own matrix. To correlate these in vitro observations with events in human diabetic retinopathy we measured integrin levels in retinal trypsin digests prepared from 10 patients with 8.2 +/- 1.6 (mean +/- SE) years of diabetes and 10 age- and sex-matched nondiabetic controls. Microvessels of diabetic patients showed increased immunostaining for beta 1 integrin (P = 0.025) when compared with control microvessels. These data show that high glucose and diabetes increase integrin expression and thus alter the interaction of vascular endothelial cells with their basement membranes in the direction of firmer cell-matrix adhesion. This could compromise the migration and replication critical to the reendothelialization process and contribute to microvascular occlusion.

Physiological aspects of vascular endothelial cell interactions in hypertension and atherosclerosis.

Hypertension causes a number of alterations of the coronary circulation which may influence the outcome of anaesthesia and surgery. These include changes in the architecture of the coronary vasculature, and impairments in coronary reserve and autoregulatory ability of the coronary microvasculature during decreases in perfusion pressure. Chronic hypertension may also alter endothelial regulation of vascular control. Many of the vasodilators used to treat hypertension may have unique effects on the coronary circulation which may become modified by chronic hypertension and cardiac hypertrophy. In this review, each of these areas are considered together with some unique aspects of endothelial/vascular smooth muscle interactions as they relate to acute and chronic hypertension, and to the pharmacological agents used in the treatment of hypertension.