Brush-Like Coatings Provide a Cloak of Invisibility to Titanium Implants.

Orthopedic implants such as knee and hip implants are one of the most important types of medical devices. Currently, the surface of the most advanced implants consists of titanium or titanium-alloys with high porosity at the bone-contacting surface leading to superior mechanical properties, excellent biocompatibility, and the capability of inducing osseointegration. However, the increased surface area of porous titanium provides a nidus for bacteria colonization leading to implant-related infections, one of the main reasons for implant failure. Here, two readily applicable titanium-coatings based on hydrophilic carboxybetaine polymers that turn the surface stealth thereby preventing bacterial adhesion and colonization are developed. These coatings are biocompatible, do not affect cell functionality, exhibit great antifouling properties, and do not cause additional inflammation during the healing process. In this way, the coatings can prevent implant-related infections, while at the same time being completely innocuous to its biological environment. Thus, these coating strategies are a promising route to enhance the biocompatibility of orthopedic implants and have a high potential for clinical use, while being easy to implement in the implant manufacturing process.

Blood Milieu in Acute Myocardial Infarction Reprograms Human Macrophages for Trauma Repair.

Acute myocardial infarction (AMI) is accompanied by a systemic trauma response that impacts the whole body, including blood. This study addresses whether macrophages, key players in trauma repair, sense and respond to these changes. For this, healthy human monocyte-derived macrophages are exposed to 20% human AMI (n = 50) or control (n = 20) serum and analyzed by transcriptional and multiparameter functional screening followed by network-guided data interpretation and drug repurposing. Results are validated in an independent cohort at functional level (n = 47 AMI, n = 25 control) and in a public dataset. AMI serum exposure results in an overt AMI signature, enriched in debris cleaning, mitosis, and immune pathways. Moreover, gene networks associated with AMI and with poor clinical prognosis in AMI are identified. Network-guided drug screening on the latter unveils prostaglandin E2 (PGE2) signaling as target for clinical intervention in detrimental macrophage imprinting during AMI trauma healing. The results demonstrate pronounced context-induced macrophage reprogramming by the AMI systemic environment, to a degree decisive for patient prognosis. This offers new opportunities for targeted intervention and optimized cardiovascular disease risk management.

Accelerating the end-to-end production of cyclic phosphate monomers with modular flow chemistry.

The biocompatibility, tunable degradability and broad functionalities of polyphosphoesters and their potential for biomedical applications have stimulated a renewed interest from Chemistry, Medicinal Chemistry and Polymer Sciences. Commercial applications of polyphosphoesters as biomaterials are still hampered because of the time and resource-intensive sourcing of their corresponding monomers, in addition to the corrosive and sensitive nature of their intermediates and by-products. Here, we present a groundbreaking challenge for sourcing the corresponding cyclic phosphate monomers by a different approach. This approach relies on the use of continuous flow technologies to intensify the end-to-end preparation of cyclic phosphate monomers with a semi-continuous modular flow platform. The applied flow technology mitigates both safety and instability issues related to the more classical production of cyclic phosphate monomers. The first flow module allows safe synthesis of a library of cyclic chlorophosphite building blocks and features in-line 31P NMR real-time monitoring. After optimization on the microfluidic scale, this first module is successfully transposed toward mesofluidic scale with a daily throughput of 1.88 kg. Downstream of the first module, a second module is present, allowing the quantitative conversion of cyclic chlorophosphites with molecular oxygen toward chlorophosphate derivatives within seconds. The two modules are concatenable with a downstream semi-batch quench of intermediate chlorophosphate with alcohols, hence affording the corresponding cyclic phosphate monomers. Such a continuous flow setup provides considerable unprecedented advantages to safely and efficiently synthesize a library of versatile high value-added cyclic phosphate monomers at large scale. These freshly produced monomers can be successfully (co)polymerized, using either batch or flow protocols, into well-defined polyphosphoesters with assessed thermal properties and cytotoxicity.

Thiol-ene Reaction: An Efficient Tool to Design Lipophilic Polyphosphoesters for Drug Delivery Systems.

Poly(ethylene glycol)-b-polyphosphoester (PEG-b-PPE) block copolymer nanoparticles are promising carriers for poorly water soluble drugs. To enhance the drug loading capacity and efficiency of such micelles, a strategy was investigated for increasing the lipophilicity of the PPE block of these PEG-b-PPE amphiphilic copolymers. A PEG-b-PPE copolymer bearing pendant vinyl groups along the PPE block was synthesized and then modified by thiol-ene click reaction with thiols bearing either a long linear alkyl chain (dodecyl) or a tocopherol moiety. Ketoconazole was used as model for hydrophobic drugs. Comparison of the drug loading with PEG-b-PPE bearing shorter pendant groups is reported evidencing the key role of the structure of the pendant group on the PPE backbone. Finally, a first evidence of the biocompatibility of these novel PEG-b-PPE copolymers was achieved by performing cytotoxicity tests. The PEG-b-PPE derived by tocopherol was evidenced as particularly promising as delivery system of poorly water-soluble drugs.

(Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration.

Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Phosphodiester Hydrogels for Cell Scaffolding and Drug Release Applications.

Given the major structural role phosphodiesters play in the organism it is surprising they have not been more widely adopted as a building block in sophisticated biomimetic hydrogels and other biomaterials. The potential benefits are substantial: phosphoester-based materials show excellent compatibility with blood, cells, and a remarkable resistance to protein adsorption that may trigger a foreign-body response. In this work, a novel class of phosphodiester-based ionic hydrogels is presented which are crosslinked via a phosphodiester moiety. The material shows good compatibility with blood, supports the growth and proliferation of tissue and presents opportunities for use as a drug release matrix as shown with fluorescent model compounds. The final gel is produced via base-induced elimination from a phosphotriester precursor, which is made by the free-radical polymerization of a phosphotriester crosslinker. This crosslinker is easily synthesized via multigram one-pot procedures out of common laboratory chemicals. Via the addition of various comonomers the properties of the final gel may be tuned leading to a wide range of novel applications for this exciting class of materials.

Synthesis of Functional Polymer Particles from Morita-Baylis-Hillman Polymerization.

Functional synthetic polymers are frequently explored for their use in the biomedical field. To fulfill the stringent demands of biodegradability and compatibility, the materials need to be versatile and tunable. Post-modification is often considered challenging for well-known degradable materials like poly(lactic acid) because of their chemical inertness. In this work a procedure is proposed to produce densely functionalized polymer particles using oligomeric precursors synthesized via the Morita-Baylis-Hillman reaction. This allows for a variety of post-modification reactions to serve bio-conjugation or tuning of the material properties. The particles are subjected to basic media and found to be degradable. Furthermore, cytotoxicity tests confirm good biocompatibility. Finally, as a proof of concept to demonstrate the versatility of the particles, post-modification reactions are carried out through the formation of imines.

Extracellular Vesicles Work as a Functional Inflammatory Mediator Between Vascular Endothelial Cells and Immune Cells.

Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.

Maintaining bovine satellite cells stemness through p38 pathway.

Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.

Direct detection of nano-scale extracellular vesicles derived from inflammation-triggered endothelial cells using surface plasmon resonance.

A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (~48.5%) and 100-300 nm (~51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV.

In vitro and in vivo evaluation of drug-eluting microspheres designed for transarterial chemoembolization therapy.

Poly(D,L-lactic acid) biodegradable microspheres, loaded with the drugs cisplatin and/or sorafenib tosylate, were prepared, characterized and studied. Degradation of the microspheres, and release of cisplatin and/or sorafenib tosylate from them, were investigated in detail. Incubation of the drug-carrying microspheres in phosphate buffered saline (pH=7.4) revealed slow degradation. Nevertheless, significant release of cisplatin and sorafenib tosylate from microspheres loaded with both drugs was apparent in vitro; this can be attributed to their porous structure. Supernatants from microspheres loaded with both drugs showed strong toxic effects on cells (i.e. endothelial cells, fibroblast cells and Renca tumor cells) and potent anti-angiogenic effect in the matrigel endothelial tube assay. In vivo anti-tumor effects of the microspheres were also observed, in a Renca tumor mouse model. The poly(D,L-lactic acid) microspheres containing both cisplatin and sorafenib tosylate revealed highest therapeutic efficacy, probably demonstrating that combined local administration of cisplatin and sorafenib tosylate synergistically inhibits tumor growth in situ. In conclusion, this study demonstrates the applicability of biodegradable poly(D,L-lactic acid) microspheres loaded with cisplatin and sorafenib tosylate for local drug delivery as well as the potential of these microspheres for future use in transarterial chemoembolization.

Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells.

Poly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA. In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein. The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood-brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p<0.05) inhibition (40%) in presence of 2-dexoxy-D-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones. As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.

Preparation and structure of drug-carrying biodegradable microspheres designed for transarterial chemoembolization therapy.

Biodegradable poly(D,L-lactic acid) drug-eluting microspheres containing anti-tumor drugs, cisplatin, and sorafenib tosylate have been prepared by the emulsion solvent evaporation method with diameter between 200 and 400 µm. Scanning electron microscopy showed that cisplatin microspheres had smooth surfaces, while sorafenib tosylate microspheres and cisplatin + sorafenib tosylate microspheres were porous at the surface and the pits of the latter were larger than those of the former. Notably, cisplatin + sorafenib tosylate microspheres had a fast drug release rate compared with microspheres containing one drug alone. In vitro cytotoxicity experiments and classical matrigel endothelial tube assay certificated the maintaining bioactivity of cisplatin and sorafenib tosylate released from the microspheres, respectively. This work provides a useful approach for the fabrication of drug-eluting beads used in transarterial chemoembolization.

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

AIMS: The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. METHODS AND RESULTS: After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CONCLUSION: CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Arginase-1 deficiency regulates arginine concentrations and NOS2-mediated NO production during endotoxemia.

RATIONALE AND OBJECTIVE: Arginase-1 is an important component of the intricate mechanism regulating arginine availability during immune responses and nitric oxide synthase (NOS) activity. In this study Arg1(fl/fl)/Tie2-Cre(tg/-) mice were developed to investigate the effect of arginase-1 related arginine depletion on NOS2- and NOS3-dependent NO production and jejunal microcirculation under resting and endotoxemic conditions, in mice lacking arginase-1 in endothelial and hematopoietic cells. METHODS AND RESULTS: Arginase-1-deficient mice as compared with control mice exhibited higher plasma arginine concentration concomitant with enhanced NO production in endothelial cells and jejunal tissue during endotoxemia. In parallel, impaired jejunal microcirculation was observed in endotoxemic conditions. Cultured bone-marrow-derived macrophages of arginase-1 deficient animals also presented a higher inflammatory response to endotoxin than control littermates. Since NOS2 competes with arginase for their common substrate arginine during endotoxemia, Nos2 deficient mice were also studied under endotoxemic conditions. As Nos2(-/-) macrophages showed an impaired inflammatory response to endotoxin compared to wild-type macrophages, NOS2 is potentially involved. A strongly reduced NO production in Arg1(fl/fl)/Tie2-Cre(tg/-) mice following infusion of the NOS2 inhibitor 1400W further implicated NOS2 in the enhanced capacity to produce NO production Arg1(fl/fl)/Tie2-Cre(tg/-) mice. CONCLUSIONS: Reduced arginase-1 activity in Arg1(fl/fl)/Tie2-Cre(tg/-) mice resulted in increased inflammatory response and NO production by NOS2, accompanied by a depressed microcirculatory flow during endotoxemia. Thus, arginase-1 deficiency facilitates a NOS2-mediated pro-inflammatory activity at the expense of NOS3-mediated endothelial relaxation.

A nontoxic additive to introduce x-ray contrast into poly(lactic acid). Implications for transient medical implants such as bioresorbable coronary vascular scaffolds.

Bioresorbable coronary vascular scaffolds are about to revolutionize the landscape of interventional cardiology. These scaffolds, consisting of a poly(L-lactic acid) interior and a poly(D,L-lactic acid) surface coating, offer a genuine alternative for metallic coronary stents. Perhaps the only remaining drawback is that monitoring during implantation is limited to two X-ray contrast points. Here, a new approach to make the biodegradable scaffolds entirely radiopaque is explored. A new contrast agent is designed and synthesized. This compound is miscible with poly(D,L-lactic acid) matrix, and nontoxic to multiple cell types. Blends of poly(D,L-lactic acid) and the contrast agent are found to be hemocompatible, noncytotoxic, and radiopaque. The data show that it is possible to manufacture fully radiopaque bioresorbable coronary vascular scaffolds. Whole-stent X-ray visibility helps interventionalists ensure that the scaffold deploys completely. This important advantage may translate into improved safety, accuracy, and clinical performance of cardiac stents.

Utilization of flax fibers for biomedical applications.

Over the past decades, a large number of animal-derived materials have been introduced for several biomedical applications. Surprisingly, the use of plant-based materials has lagged behind. To study the feasibility of plant-derived biomedical materials, we chose flax (Linum usitatissimum). Flax fibers possess excellent physical-mechanical properties, are nonbiodegradable, and there is extensive know-how on weaving/knitting of them. One area where they could be useful is as implantable mesh structures in surgery, in particular for the repair of incisional hernias of the abdominal wall. Starting with a bleached flax thread, a prototype mesh was specifically knitted for this study, and its cytocompatibility was studied in vitro and in vivo. The experimental data revealed that application of flax in surgery first requires a robust method to remove endotoxins and purify the flax fiber. Such a method was developed, and purified meshes did not cause loss of cell viability in vitro. In addition, endotoxins determined using limulus amebocyte lysate test were at acceptable levels. In vivo, the flax meshes showed only mild inflammation, comparable to commercial polypropylene meshes. This study revealed that plant-derived biomaterials can provide a new class of implantable materials that could be used as surgical meshes or for other biomedical applications.

Specific visualization of nitric oxide in the vasculature with two-photon microscopy using a copper based fluorescent probe.

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.