The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water-permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion-assisted decellularization.

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis.

The aim of this study is to establish the expression of ATP-sensitive potassium channels(KATP) in human umbilical arteries with severe pre-eclampsia. Real-time quantitative PCR and western blotting were used to detect the mRNA and protein expression levels of KATP channel subunits Kir6.1 and SUR2B in human umbilical arteries from normal pregnant and those with severe pre-eclampsia, early onset severe pre-eclampsia and late onset severe pre-eclampsia. The mRNA and protein levels of SUR2B in the severe pre-eclampsia group were lower than those in the normal group (P < 0.001), and the expression of Kir6.1 was not statistically significant between the two groups (P > 0.05). The mRNA and protein levels of SUR2B in early onset severe pre-eclampsia group were lower than those in late onset severe pre-eclampsia group (P < 0.001). There was no significant difference in expression of Kir6.1 between the two groups (P > 0.05). The mRNA and protein expression levels of SUR2B in pregnant women with severe pre-eclampsia were lower than those in normal pregnant women, suggesting that the expression of the SUR2B of the KATP channel may be related to the occurrence and development of severe pre-eclampsia. Compared with late onset severe pre-eclampsia, the mRNA and protein expression levels of SUR2B were lower in the umbilical arteries of women with early onset severe pre-eclampsia, suggesting that the occurrence time of severe pre-eclampsia may be related to the extent reduced expression of the SUR2B of the KATP channel.

High levels of proteins called proteoglycans in the walls of umbilical arteries enable these arteries to close rapidly after birth and thus prevent blood loss in newborns.

Human mesenchymal stem cells derived from the umbilical cord (UC) are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs), perivascular stem cells derived from umbilical vein (UCV-PSCs), and mesenchymal stem cells derived from Wharton's jelly (WJ-MSCs). These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P < 0.05). Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P < 0.01). Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1), which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases.

Targeted drug delivery to diseased vasculature, such as atherosclerotic lesions, is a multistep process, which is based on the transport of drug carriers to a selected region and their deposition at the desired destination. Current modeling approaches, including microfluidics and animal models, fail to accurately simulate this multi-scale process in human arteries, where blood flow is dominant. Here we study particle deposition in endothelialized 3D reconstructed models of the human carotid bifurcation under physiological hemodyamic conditions. Our results showed that particle localization is highly dependent on vessel geometry and local flow features. Additionally, while strongly adhesive particles tend to adhere more profoundly at high-shear regions, associated with athero-thrombosis, enhanced deposition at vascular flow regions, associated with inflammation and plaque accumulation, e.g., recirculation flows, can be achieved using weakly adhesive particles. Moreover, pulsatile flow as well as presence of blood cells significantly reduce particle adhesion and affect their deposition pattern. These findings highlight the key role of vessel geometry, hemodynamics and particle characteristics in the optimizing vascular targeting nano-carriers.

Animal disease models contribute to a better understanding of the pathogenic mechanisms of human and animal diseases and help develop treatments for them. Ligation of the rat iliac arteries is performed to reproduce erectile dysfunction and peripheral arterial disease. Although knowledge of the ramification of branches from the rat iliac artery is important to perform such surgery, descriptions in previous studies are insufficient. Therefore, 17 male and 18 female Wistar rats were observed to elucidate the detailed ramification patterns of branches from the iliac arteries with the latex injection method. The iliac arteries branched off the umbilical, cranial gluteal, lateral and medial circumflex femoral, external pudendal, and caudal epigastric arteries, and the common trunk of the caudal gluteal and internal pudendal arteries. The branching pattern of the umbilical, cranial and caudal gluteal, and internal pudendal arteries varied greatly and was categorized as Types 1 to 3 based on the number of branching levels along the proximodistal axis of the iliac arteries. Based on the same criteria, the ramification patterns of the lateral and medial circumflex femoral arteries were also divided into Groups 1 and 2. The external pudendal and caudal epigastric arteries originated from the external iliac artery mainly as a common trunk or less frequently as independent arteries in this order. The detailed branching patterns of the rat iliac arteries elucidated in the present study are beneficial for the refinement of surgical procedures.

The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries.

Di(2-etilhexil) phthalate (DEHP) is a compound used in plastic materials, which has endocrine disrupting properties. The human DEHP exposure depend on the use of plastics in toys, medical devices and food and beverage containers. The DEHP effects were studied in some physiological systems; nevertheless, the actions in human arteries were never described. We analysed the DEHP effect on endothelium denuded human umbilical artery (HUA), an important artery to ensure gases and nutrients exchange with fetus. We assessed DEHP short-term effects on contractility, occurring few minutes after DEHP is in contact with HUA in the organ bath receptacles. The long-term effects on HUA, observed after 24 h in presence of DEHP, were assessed in the organ bath system, and also through the analysis of receptors expression (5-HT2A and H1) and of cellular viability, by using HUA smooth muscle cells. DEHP (1 nM-100 μM) induced a short-term relaxing effect on HUA contracted by 5-HT, histamine or KCl. DEHP long-term exposure of arteries (1 nM, 10 μM and 100 μM) reduced its own relaxant effect on HUA contracted by 5-HT and histamine and, precisely, 24 h exposure to DEHP 1 nM reverted the relaxant effect on 5-HT contractility. Long-term exposure at more than 10 nM of DEHP decreased 5HT2A receptors expression. In conclusion, DEHP short-term exposition elicit vasodilation of HUA contracted by different agents. DEHP long-term exposition reduced the expression of 5HT2A receptors. The DEHP long-term exposition decrease the short-term relaxant effect and, at low concentrations can increase the contractile effect of 5-HT.

Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology.

In vascular tissue engineering strategies, the addition of vascular-specific extracellular matrix (ECM) components may better mimic the in vivo microenvironment and potentially enhance cell-matrix interactions and subsequent tissue growth. For this purpose, the exact composition of the human vascular ECM first needs to be fully characterized. Most research has focused on characterizing ECM components in mature vascular tissue; however, the developing fetal ECM matches the active environment required in vascular tissue engineering more closely. Consequently, we characterized the ECM protein composition of active (fetal) and quiescent (mature) renal arteries using a proteome analysis of decellularized tissue. The obtained human fetal renal artery ECM proteome dataset contains higher levels of 15 ECM proteins versus the mature renal artery ECM proteome, whereas 16 ECM proteins showed higher levels in the mature tissue compared to fetal. Elastic ECM proteins EMILIN1 and FBN1 are significantly enriched in fetal renal arteries and are mainly produced by cells of mesenchymal origin. We functionally tested the role of EMILIN1 and FBN1 by anchoring the ECM secreted by vascular smooth muscle cells (SMCs) to glass coverslips. This ECM layer was depleted from either EMILIN1 or FBN1 by using siRNA targeting of the SMCs. Cultured endothelial cells (ECs) on this modified ECM layer showed alterations on the transcriptome level of multiple pathways, especially the Rho GTPase controlled pathways. However, no significant alterations in adhesion, migration or proliferation were observed when ECs were cultured on EMILIN1- or FNB1-deficient ECM. To conclude, the proteome analysis identified unique ECM proteins involved in the embryonic development of renal arteries. Alterations in transcriptome levels of ECs cultured on EMILIN1- or FBN1-deficient ECM showed that these candidate proteins could affect the endothelial (regenerative) response.

Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant's of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.

The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.

Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons.

Inflammation and hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) is considered the primary pathological feature of pulmonary hypertension (PH). The present study determined that mesenchymal stem cells (MSCs) suppress the expression of calcineurin (CaN) and nuclear factor of activated T-cells (NFAT) in the pulmonary arteries of rats, and this may exert a therapeutic effect on PH. The potential therapeutic effects of MSCs on PH were assessed via the transplantation of human umbilical cord-derived MSCs, which were cultured in serum-free medium, into a monocrotaline (MCT)-induced PH rat model. Subsequently, the expression levels of tumor necrosis factor (TNF)-α in lung tissue and plasma, and of CaN and NFATc2 in pulmonary arteries were assessed. In the rat model of MCT-induced PH, investigated in the present study, TNF-α expression levels were detected in the lung tissue, and the levels of TNF-α in the plasma were increased. Furthermore, in addition to hemodynamic changes and the evident medial hypertrophy of the pulmonary muscular arterioles, CaN and NFATc2 expression levels were significantly upregulated in the pulmonary arteries. In the present study, the transplantation of MSCs, cultured in serum-free medium, decreased the levels of TNF-α in the lung tissue and plasma of rats, and downregulated CaN and NFATc2 expression in the pulmonary arteries. Furthermore, hemodynamic abnormalities and medial hypertrophy of the pulmonary muscular arterioles were notably improved. Therefore, the results of the present study may suggest that the administration of MSCs in PH may suppress the production of TNF-α, and downregulate the expression of CaN and NFATc2 in pulmonary arteries, which may provide an effective treatment for PH by suppressing the pathological proliferation of PASMCs.

Menopause causes arterial senescence and atherosclerotic development through decrease of estrogen. Recently, histone deacetylase SIRT1 has been reported to have protective effects against arterial senescence and atherosclerosis. However, the relationship between estrogen and SIRT1 in the context of menopause-induced arterial senescence is not well understood. The present study aims to investigate whether SIRT1 is involved in the etiology of menopause-induced arterial senescence and atherosclerotic development.

Bee propolis is a mixture of plant resins and bee secretions. While bioactivity of honeybee propolis has been reported previously, information is limited on propolis from Australian stingless bees (Tetragonula carbonaria). The aim of this study was to investigate possible vasomodulatory effects of propolis in KCl-precontracted porcine coronary arteries using an ex vivo tissue bath assay. Polar extracts of propolis produced a dose-dependent relaxant response (EC50=44.7±7.0 μg/ml), which was unaffected by endothelial denudation, suggesting a direct effect on smooth muscle. Propolis markedly attenuated a contractile response to Ca(2+) in vessels that were depolarised with 60 mM KCl, in Ca(2+)-free Krebs solution. Propolis (160 µg/ml) reduced vascular tone in KCl pre-contracted vessels to near-baseline levels over 90 min, and this effect was partially reversible with 6 h washout. Some loss in membrane integrity, but no loss in mitochondrial function was detected after 90 min exposure of human cultured umbilical vein endothelial cells to 160 µg/ml propolis. We conclude that Australian stingless bee (T. carbonaria) propolis relaxes porcine coronary artery in an endothelial-independent manner that involves inhibition of voltage-gated Ca(2+) channels. This effect is partially and slowly reversible upon washout. Further studies are required to determine the therapeutic potential of Australian stingless bee propolis for conditions in which vascular supply is compromised.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.