Selective adhesion and growth of vascular endothelial cells on bioactive peptide nanofiber functionalized stainless steel surface

Metal-based scaffolds such as stents are the most preferred treatment methods for coronary artery disease. However, impaired endothelialization on the luminal surface of the stents is a major limitation occasionally leading to catastrophic consequences in the long term. Coating the stent surface with relevant bioactive molecules is considered to aid in recovery of endothelium around the wound site. However, this strategy remains challenging due to restrictions in availability of proper bioactive signals that will selectively promote growth of endothelium and the lack of convenience for immobilization of such signaling molecules on the metal surface. In this study, we developed self-assembled peptide nanofibers that mimic the native endothelium extracellular matrix and that are securely immobilized on stainless steel surface through mussel-inspired adhesion mechanism. We synthesized Dopa-conjugated peptide amphiphile and REDV-conjugated peptide amphiphile that are self-assembled at physiological pH. We report that Dopa conjugation enabled nanofiber coating on stainless steel surface, which is the most widely used backbone of the current stents. REDV functionalization provided selective growth of endothelial cells on the stainless steel surface. Our results revealed that adhesion, spreading, viability and proliferation rate of vascular endothelial cells are remarkably enhanced on peptide nanofiber coated stainless steel surface compared to uncoated surface. On the other hand, although vascular smooth muscle cells exhibited comparable adhesion and spreading profile on peptide nanofibers, their viability and proliferation significantly decreased. Our design strategy for surface bio-functionalization created a favorable microenvironment to promote endothelial cell growth on stainless steel surface, thereby providing an efficient platform for bioactive stent development for long term treatment of cardiovascular diseases.     

Zwitterionic polycarboxybetaine coating functionalized with REDV peptide to improve selectivity for endothelial cells

Surface immobilization of bioactive molecules has been a promising strategy to develop in situ endothelialization for cardiovascular implants. With the aim to construct endothelial cell specific coating with low fouling property, zwitterionic carboxybetaine methacrylate and butyl methacrylate were copolymerized as coating materials, spin-coated onto substrates, and immobilized with endothelial cell selective peptide Arg-Glu-Asp-Val (REDV) through functionalization of carboxy groups in carboxybetaine by NHS/EDC chemistry. Experimental results proved that carboxybetaine-REDV coating maintained desirable antifouling ability and fine hemocompatibility. Separate culture and coculture of HUVECs (human umbilical vein endothelial cells) with HUASMCs (human umbilical artery smooth muscle cells) showed that the coating was able to enhance the competitive growth of endothelial cells while limiting the adhesion, proliferation, and migration of smooth muscle cells. The existence of zwitterionic carboxybetaine helps to screen undesirable adsorption of platelets, and its nonspecific resistance to smooth muscle cells contributes to the realization of endothelial cell selectivity.     

Polysaccharide-based biomaterials with on-demand nitric oxide releasing property regulated by enzyme catalysis

The regulatory role of nitric oxide (NO) in cell signaling has been well recognized. Clinically, NO deficiency is known to be associated with severe vascular disorders, especially in patients with long-term diabetes. Exogenous compensation of NO is a promising therapeutic strategy, although the lack of stable NO compounds often lead to unsatisfactory clinical outcomes. In the present study, we report a stable comb-shaped polymer (CS–NO) using glycosylated NO compound as pendent chains and chitosan (CS) as backbone for controlled NO release. The on-demand release of NO is achieved by controlling the decomposition process of the CS–NO polymer, which is blocked by galactose and only occurs in the presence of glycosidase, making the NO releasing kinetic closely correlate with the glycosidase concentration. In addition, due to its high stability, the CS–NO polymers can also be processed into supportive membrane or injectable hydrogel, further demonstrating its clinical potential. Indeed, we report that the NO-releasing membrane inhibited platelet adhesion, prolonged activated partial thromboplastin time (APTT) as shown in the platelet-rich-plasma (PRP) assay. We also observe enhanced human umbilical vein endothelial cell growth yet suppressed vascular smooth muscle cell proliferation on the NO-contained membrane in vitro. Furthermore, in vivo administration of CS–NO solution significantly enhanced angiogenesis in diabetic mice with hind-limb ischemia. Protective effect of CS–NO was also observed against limb necrosis. Given the physiological importance of NO, the CS–NO polymer may be considered a promising option in therapeutic development against vascular disorders and diabetic feet.     

Inhibition of blood vessel formation by a chondrocyte-derived extracellular matrix

In this study, the chondrocyte-derived extracellular matrix (CECM) was evaluated for its activity to inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) and rabbit chondrocytes were plated on a bio-membrane made of CECM or human amniotic membrane (HAM). The adhesion, proliferation, and tube formation activity of HUVECs and chondrocytes were examined. The CECM and HAM powders were then mixed individually in Matrigel and injected subcutaneously into nude mice to examine vessel invasion in vivo after 1 week. Finally, a rabbit model of corneal neovascularization (NV) was induced by 3-point sutures in the upper cornea, and CECM and HAM membranes were implanted onto the corneal surface at day 5 after suture injury. The rabbits were sacrificed at 7 days after transplantation and the histopathological analysis was performed. The adhesion and proliferation of HUVECs were more efficient on the HAM than on the CECM membrane. However, chondrocytes on each membrane showed an opposite result being more efficient on the CECM membrane. The vessel invasion in vivo also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CECM. In the rabbit NV model, CECM efficiently inhibited the neovessels formation and histological remodeling in the injured cornea. In summary, our findings suggest that CECM, an integral cartilage ECM composite, shows an inhibitory effect on vessel invasion both in vitro and in vivo, and could be a useful tool in a variety of biological and therapeutic applications including the prevention of neovascularization after cornea injury.     

Design of a Novel Apparatus to Study Nitric Oxide (NO) Inhibition of Platelet Adhesion

Nitric oxide (NO) is a simple biological molecule which inhibits adhesion and aggregation of platelets. A novel NO delivery device has been developed to quantitatively study the effects of NO concentration and flux on the adhesion of platelets to a surface. The slit-flow device is lined with a protein-coated membrane through which NO gas permeates into a perfusing platelet suspension. A model predicting spatial NO concentrations and fluxes within the flow slit was validated. At a wall shear rate of 250s^-1, platelet adhesion was inhibited 87% relative to controls for exposures as low as 0.1 ppm NO. Corresponding model predictions of the aqueous NO concentration and fluxes at the surface were 0.15 nM, and between 0.5 and 1.1 nanomoles cm^-2 s_-1, respectively. Endo-thelial cells, which release NO to inhibit platelet adhesion in vivo, generate NO at an estimated flux similar to the above values. At a NO exposure of 0.02 ppm, platelet inhibition was only 10%. The delivery device is useful for other studies in which a knowledge of the spatial NO fluxes or concentrations is desired. Knowledge of these fluxes or concentrations is beneficial towards the design of biomaterials incorporating NO to inhibit platelet adhesion. © 1998 Biomedical Engineering Society. PAC98: 8745Hw, 8722Fy, 8780+s, 8710+e, 8380Lz     

Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents

Coronary stents that are developed for use with balloon angioplasty are known to cause acute occlusion and long-term stenosis. It is likely that a controlled release of drugs at the site of stent implantation might inhibit the proliferation of vascular smooth muscle cells (VSMC) and reduce restenosis. However, if the drug is necrotic and affects cell survival near the implant, it may interrupt the local tissue regeneration. Different methods have been used for the immobilization of drugs with stents to get an effective concentration that inhibits cell proliferation. The objective of this study is to assess the effectiveness of Paclitaxel-loaded stents by immobilization with a biodegradable polymer, to inhibit cell proliferation. The cells used for the evaluation are human umbilical vein endothelial cells (HUVEC) and the proliferation rate of these cells on the drug-coated stent is compared against an uncoated stent for a 72-h period. Evaluations were also made to differentiate between cell apoptosis and necrosis to prove that the drug released is not deleterious to the surrounding tissue.While a similar initial cell adhesion is observed in bare and coated stents, the proliferation of HUVEC is negligible when grown on a drug-coated stent (p < 0.001). By specific staining techniques, the cells on the drug-coated stents are found to be apoptotic and not necrotic, throughout the evaluation period. In vitro leukocyte adhesion and platelet deposition on the drug-coated stents are found to be low when they are exposed to human blood and platelet-rich plasma (PRP), suggesting that the coated stents may not be thrombogenic in vivo. Therefore, drug coating of stents using the described technique may have a considerable promise for the prevention of neointimal proliferation, restenosis, and associated failure of angioplasty.     

Electropolymerization of dopamine for surface modification of complex-shaped cardiovascular stents

Inspired by the adhesion strategy of marine mussels, self-polymerization of dopamine under alkaline condition has been proven to be a simple and effective method for surface modification of biomaterials. However, this method still has many drawbacks, such as the use of alkaline aqueous medium, low poly(dopamine) deposition rate, and inefficient utilization of dopamine, which greatly hinder its practical application. In the present study, we demonstrate that electropolymerization of dopamine is a facile and versatile approach to surface tailoring of metallic cardiovascular stents, such as small and complex-shaped coronary stent. Electropolymerization of dopamine leads to the formation of a continuous and smooth electropolymerized poly(dopamine) (ePDA) coating on the substrate surface. This electrochemical method exhibits a higher deposition rate and is more efficient in dopamine utilization compared with the typical self-polymerization method. The ePDA coating facilitates the immobilization of biomolecules onto substrates to engineer biomimetic microenvironments. In vitro and in vivo experiments demonstrate that ePDA coating functionalized with vascular endothelial growth factor can greatly enhance the desired cellular responses of endothelial cells and prevent the neointima formation after stent implantation. The proposed methodology may find applications in the area of metallic surface engineering, especially for the cardiovascular stents and potentially all biomedical devices with electroconductive surface as well.     

In vitro study of cell-promoting multiple-armed peptides

The purpose of this study was to compare the effectiveness of several linear and branch cell-binding peptides to promote cell growth in prosthetic vascular grafts. In this in vitro study, the peptides were covalently immobilized onto expanded polytetrafluoroethylene (ePTFE) vascular grafts. Cell-growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). Linear peptides (P15 and P15') and multiple-armed peptides (MAP4-I and MAP4-II) were covalently bonded onto ePTFE grafts by an atmospheric plasma coating method. X-ray photoelectron spectroscopy and amino acid analysis were used to analyze the surface characteristics of the peptide-coated samples. Cell adhesion, proliferation, and morphology were evaluated by culturing HUVECs and HUASMCs onto the surfaces of different samples: ePTFE control, chemically activated ePTFE, P15-coated ePTFE, and MAP4-coated ePTFE. The cell culture experiments were repeated several times to obtain statistically reliable cell-growth data. Cell-growth data were statistically analyzed by the two-way statistical analysis of variance. The study showed that multiple-armed MAP4 peptides were significantly more effective in promoting endothelial cells than the structurally similar linear P15 peptides. There were 800% more HUVECs proliferated on the MAP4-coated ePTFE samples compared with the ePTFE control. MAP4 peptides were 80% more effective for promoting HUVECs than P15 peptides. In contrast, MAP4 peptides were significantly less effective for promoting HUASMCs than HUVECs. There were only about 100% more HUASMCs proliferated on the MAP4-coated ePTFE samples compared with the ePTFE control. MAP4 and P15 peptides had similar cell-promoting characteristics for SMCs.     

Transport Limitations of Nitric Oxide Inhibition of Platelet Aggregation under Flow

Nitric oxide (NO) inhibits platelet aggregation at and near the site of a vascular injury by upregulation of cyclic guanosine monophosphate, which reduces the dimerization of the integrin α _IIb β ₃. The magnitude of NO flux from the vessel wall and the NO concentration that is necessary to inhibit platelet aggregation under physiological flow conditions is unknown. In this study, a NO releasing polymer, diazeniumdiolated dibutylhexanediamine, was integrated into a microfluidic flow assay to determine the relationship between NO wall flux and collagen mediated platelet adhesion, activation and aggregation. A NO flux equal to or greater than 2.5 × 10^?10 mol cm^?2 min^?1 was found to abrogate aggregation, but not initial platelet adhesion, on collagen at 200 and 500 s^?1 as effectively as the α _IIb β ₃ antagonist abciximab. The dynamic range of NO fluxes found to induce measurable inhibition of platelet aggregation spanned from 0.33 × 10^?10 to 2.5 × 10^?10 mol cm^?2 min^?1 at 200–500 s^?1. These fluxes correspond to near-wall NO concentrations of 3–90 nM based on a computational model of NO transport. The model predicts that NO concentration in the platelet rich layer near the wall is kinetically limited, while NO penetration into the lumen is mass transfer limited.     

In-vitro assays of polymer-coated stents eluting platelet glycoprotein IIb/IIIa receptor monoclonal antibody

The monoclonal antibody (mAb) to the platelet glycoprotein (GP) IIb/IIIa receptor has potent antiplatelet and antithrombotic characteristics shown to reduce thrombus-related major complications after coronary angioplasty. This mAb can be incorporated in drug-eluting stents capable of releasing single or multiple bioactive agents into the bloodstream and surrounding tissues. Stents eluting the monoclonal mouse anti-human platelet glycoprotein IIb/IIIa antibody SZ-262 were tested for their effectiveness in improving the blood compatibility and the antithrombotic characteristics by immunofluorescence and scanning electron microscopy (SEM). The SEM results convincingly demonstrated that the surface of the mAb eluting-stents was completely free of platelet uptake without any sign of cellular debris or proteinaceous deposits, compared with controls. The deformation index of platelets on the L-polylactic acid (L-PLA) coated stents were higher than bare Nitinol intravascular stents, as shown by SEM images. Monoclonal antibody to the platelet GP IIb/IIIa receptor, when eluting from L-PLA polymer-coated stents, effectively inhibits platelet aggregation in the stent microenvironment, thus demonstrating a potential capacity of reducing thrombosis, improving blood flow and arterial patency rates, and inhibiting cyclic blood flow variations. These results highlight the possibility of such monoclonal antibody-eluting stents to reduce or possibly eliminate thrombosis and in-stent restenosis.     

Can we extrapolate the outcomes of in vitro studies on murine endothelium to studies of human platelet-endothelium interactions? A technical note

Introduction

Interactions between vascular endothelium and blood platelets play a crucial role in cardiovascular diseases. Ex vitro models which use endothelial cells and platelets were the essential tools to investigate these interactions and their impact on haemostasis. The impaired interplay between vascular endothelium, blood platelets and leukocytes is believed to contribute to the development of cardiovascular disease. In this study we compared the ability of human (HUVECs) and murine (HECa10) endothelial cells to inhibit human platelet function and reactivity under in vitro conditions.

Material and methods

The aliquots of platelet-rich plasma obtained from 20 healthy donors were incubated with murine endothelial cell line HECa10 or human umbilical vein endothelial cells (HUVECs) (10 min, 37°C) prior to agonizing platelets with 5 µM ADP and monitoring platelet reactivity for 10 min using optical aggregation.

Results

Significant reduction in ADP-induced platelet aggregation in the presence of endothelial cell cultures remained independent of cell count. HUVECs appeared much more effective in the inhibition of platelet aggregation compared to HECa10 (35.2 ±2.3 AU vs. 43.7 ±2.0 AU, p= 0.025).

Conclusions

HECa10 cells have much lower potential to inhibit platelet aggregation than HUVECs. This implies that these two cell lines may not be freely used interchangeably in in vitro experiments. These findings clearly indicate that the outcomes of in vitro studies performed with murine EC lines cannot be unreservedly extrapolated to human platelet-endothelium interactions.     

The role of surface chemistry in determining in vivo biodistribution and toxicity of CdSe/ZnS core–shell quantum dots

To examine the effect of surface chemistry and surface charge on in vivo biodistribution and toxicity of CdSe/ZnS core–shell quantum dots (QDs), QDs with positive, negative, or PEG coating are used in this study for in vivo evaluation in a mouse model. The results suggest that QDs coated with cationic polydiallyldimethylammonium chloride (PDDA) preferentially deposit in the lung other than in the liver, while the negative and PEGylated QDs render abundant accumulation in the liver. At higher doses positive QDs with PDDA coating show severe acute toxicity due to pulmonary embolism. Independent of their surface coatings, all QDs cause injuries in specific tissues like liver, spleen, lung, and kidney, after acute and long-term exposure, and the degree of injuries is dominated by their surface properties. For the positively charged QDs, the acute phase toxicity is primarily contributed by the coating material PDDA, while coating on QDs may amplify both in vitro and in vivo toxicity of PDDA. PEGylated QDs display the slightest chronic injuries in the long-term toxicity examination in comparison to positive or negative ones.     

Immobilization of selenocystamine on TiO2 surfaces for in situ catalytic generation of nitric oxide and potential application in intravascular stents

Immobilization of selenocystamine on TiO(2) film deposited on silicon wafer and 316 stainless steel stents for catalytic generation of nitric oxide was described. Polydopamine was used as the linker for immobilization of selenocystamine to the TiO(2) surface. In vitro stability of the immobilized selenocystamine was investigated and the result shows surface selenium loss occurs mostly in the first four weeks. The selenocystamine immobilized surface possesses glutathione peroxidase (GPx) activity, and the activity increases with the amount of grafted polydopamine. Such selenocystamine immobilized surfaces show the ability of catalytically decomposing endogenous S-nitrosothiols (RSNO), generating NO; thus the surface displays the ability to inhibit collagen-induced platelet acitivation and aggregation. Additionally, smooth muscle cells are inhibited from adhering to the selenocystamine immobilized sample when RSNO is added to the culture media. ELISA analysis reveals that cGMP in both platelets and smooth muscle cells significantly increases with NO release on selenocystamine immobilized samples. Two months in vivo results show that selenocystamine immobilized stents are endothelialized, and show significant anti-proliferation properties, indicating that this is a favorable method for potential application in vascular stents.     

Cytocompatibility,osseointegration, and bioactivity of three-dimensional porous and nanostructured network on polyetheretherketone

Porous biomaterials with the proper three-dimensional (3D) surface network can enhance biological functionalities especially in tissue engineering, but it has been difficult to accomplish this on an important biopolymer, polyetheretherketone (PEEK), due to its inherent chemical inertness. In this study, a 3D porous and nanostructured network with bio-functional groups is produced on PEEK by sulfonation and subsequent water immersion. Two kinds of sulfonation-treated PEEK (SPEEK) samples, SPEEK-W (water immersion and rinsing after sulfonation) and SPEEK-WA (SPEEK-W with further acetone rinsing) are prepared. The surface characteristics, in vitro cellular behavior, in vivo osseointegration, and apatite-forming ability are systematically investigated by X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, cell adhesion and cell proliferation assay, real-time RT-PCR analysis, micro-CT evaluation, push-out tests, and immersion tests. SPEEK-WA induces pre-osteoblast functions including initial cell adhesion, proliferation, and osteogenic differentiation in vitro as well as substantially enhanced osseointegration and bone-implant bonding strength in vivo and apatite-forming ability. Although SPEEK-W has a similar surface morphology and chemical composition as SPEEK-WA, its cytocompatibility is inferior due to residual sulfuric acid. Our results reveal that the pre-osteoblast functions, bone growth, and apatite formation on the SPEEK surfaces are affected by many factors, including positive effects introduced by the 3D porous structure and SO₃H groups as well as negative ones due to the low pH environment. Surface functionalization broadens the use of PEEK in orthopedic implants.     

Effect of allicor on platelet aggregationin vitro andex vivo

An extract of garlic powder in isotonic phosphate buffer and adjoen (bioactive compound isolated from garlic powder) suppress human blood platelet aggregation induced by ADP and arachidonic acidin vitro. Adjoen more effectively than aspirin inhibits ADP-induced platelet aggregation but is inferior to aspirin if platelet aggregation is induced by arachidonic acid.Ex vivo oral intake of one Allicor tablet significantly decreases rabbit platelet aggregation induced with ADP. It is suggested that long-acting garlic powder tablets prevent thromboembolic complications and are recommended for correcting hemostasis parameters in patients with atherosclerotic involvement of blood vessels. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 7, pp. 79–100, July, 1997     

A novel CX3CR1 antagonist eluting stent reduces stenosis by targeting inflammation

We evaluated the therapeutic efficacy of a novel drug eluting stent (DES) inhibiting inflammation and smooth muscle cell (SMC) proliferation. We identified CX₃CR1 as a targetable receptor for prevention of monocyte adhesion and inflammation and in-stent neointimal hyperplasia without interfering with stent re-endothelization. Efficacy of AZ12201182 (AZ1220), a CX₃CR1 antagonist was evaluated in inhibition of monocyte attachment in vitro. A prototype AZ1220 eluting PLGA-based polymer coated stent developed with an optimal elution profile and dose of 1 μM/stent was tested over 4 weeks in a porcine model of coronary artery stenting. Polymer coated stents without AZ1220 and bare metal stents were used as controls. AZ1220 inhibited monocyte attachment to CX₃CL1 in a dose dependent manner. AZ1220 eluted from polymer coated stents in an ex vivo flow system retained bioactivity in inhibiting monocyte attachment to CX₃CL1. At 4 weeks following deployment, AZ1220 eluting stents significantly reduced (∼60%) in-stent stenosis compared to both bare metal and polymer only coated stents and markedly reduced peri-stent inflammation and monocyte/macrophage accumulation without affecting re-endothelization. Anti-CX₃CR1 drug eluting stents potently inhibited in-stent stenosis and may offer an alternative to mTOR targeting by current DES, specifically inhibiting polymer-induced inflammatory response and SMC proliferation, while retaining an equivalent re-endothelization response to bare metal stents.     

The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan

Controlled neo-endothelialisation is critical to the patency of small diameter vascular grafts. Endothelialisation and platelet adhesion to purified endothelial cell-derived perlecan, the major heparan sulfate (HS) proteoglycan in basement membranes, were investigated using in vivo and in vitro assays. Expanded polytetrafluoroethylene (ePTFE) vascular grafts were coated with perlecan and tested in an ovine carotid interposition model for a period of 6 weeks and assessed using light and scanning microscopy. Enhanced endothelial cell growth and reduced platelet adhesion were observed on the perlecan coated grafts when compared to uncoated controls implanted in the same sheep (n = 5). Perlecan was also found to stimulate endothelial cell proliferation in vitro over a period of 6 days in the presence of plasma proteins and fibroblastic growth factor 2 (FGF-2), however in the absence of FGF-2 endothelial cell growth could not be maintained during this period. Perlecan was found to be anti-adhesive for platelets, however after removal of the HS chains attached to perlecan, platelet adhesion and aggregation were supported. These results suggest a role for HS chains of perlecan in improving graft patency by selectively promoting endothelial cell proliferation while modulating platelet adhesion.     

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678–2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.     

Vascular biosynthesis of nitric oxide: effect on hemostasis and fibrinolysis

Nitric oxide (NO) is a multifunctional effector molecule that plays a central role in the regulation of vascular homeostasis. NO is synthesized from L-arginine by a family of enzymes called NO synthases. The principal source of NO in the vascular system of healthy mammals is the constitutively expressed NO synthase in endothelial cells. The basal endothelial formation of NO can be increased by receptor-dependent agonists (i.e., bradykinin) in a calcium-calmodulin-dependent manner, and also by physical forces (i.e., shear stress), predominantly without changes in the intracellular concentration of free calcium. Nitric oxide can diffuse toward the blood vessel wall where the major target is the smooth muscle cell. NO regulates vascular tone, and the free radical is also a potent inhibitor of smooth muscle cell proliferation, migration and synthesis of extracellular matrix proteins. NO can also diffuse toward the lumen of the blood vessel where it helps maintain blood fluidity. NO inhibits platelets' and leucocytes' adhesion to endothelial cells. In addition, NO inhibits platelet aggregation and facilitates the dissolution of small platelet aggregates. However, the regulatory action of NO on blood cells is most likely limited to the luminal surface of endothelial cells since NO is rapidly scavenged by hemoglobin in erythrocytes and inactivated by oxygen-derived radicals such as superoxide anions. NO can also affect the fibrinolytic activity by regulating the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1. The crucial role of vascular NO in the control of blood fluidity has been demonstrated by the regulation of the bleeding time in humans.     

Inhibition of tumor cell-platelet interactions and tumor metastasis by the calcium channel blocker,nimodipine

Nimodipine, a dihydropyridine calcium channel blocker, was evaluated in vitro for its ability to inhibit platelet aggregation induced by B 16 amelanotic melanoma (B16a) and Walker 256 carcinosarcoma (W256) cells, and for its ability to inhibit platelet-enhanced B16a and W256 adhesion to rat microvascular endothelial cells. Nimodipine produced a dose-dependent inhibition of tumor-cell-induced platelet aggregation (TCIPA). Platelets enhanced tumor cell adhesion to endothelium both in the presence and absence of overt platelet aggregation. However, the greatest enhancement of adhesion occurred under aggregatory conditions. Nimodipine at a dose of 40 µg/ml inhibited platelet-enhanced adhesion to endothelium under aggregatory and nonaggregatory conditions. Nimodipine was tested in vivo for its ability to inhibit both experimental and spontaneous metastasis. Nimodipine produced a 46 per cent inhibition of lung colony formation at a dose of 5 mg/kg body-weight. Over a dose range of 0·1–80 mg/kg, nimodipine produced a significant dose-dependent inhibition in the formation of lung metastases from a subcutaneous tumor. The in vitro results demonstrate that a dihydropyridine calcium channel blocker can inhibit tumor cell-platelet-endothelial cell interactions. The in vivo results suggest that these compounds may be a new class of antimetastatic agent.