Demonstration of ultrasound-mediated therapeutic delivery of fibrin-targeted pioglitazone-loaded echogenic liposomes into the arterial bed for attenuation of peri-stent restenosis.

Peri-stent restenosis following stent implantation is a major clinical problem. We have previously demonstrated that ultrasound-facilitated liposomal delivery of pioglitazone (PGN) to the arterial wall attenuated in-stent restenosis. To evaluate ultrasound mediated arterial delivery, in Yucatan miniswine, balloon inflations were performed in the carotid and subclavian arteries to simulate stent implantation and induce fibrin formation. The fibrin-binding peptide, GPRPPGGGC, was conjugated to echogenic liposomes (ELIP) containing dinitrophenyl-L-alanine-labelled pioglitazone (DNP-PGN) for targeting purposes. After pre-treating the arteries with nitroglycerine, fibrin-binding peptide-conjugated PGN-loaded ELIP (PAFb-DNP-PGN-ELIP also termed atheroglitatide) were delivered to the injured arteries via an endovascular catheter with an ultrasound core, either with or without ultrasound application (EKOSTM Endovascular System, Boston Scientific). In arteries treated with atheroglitatide, there was substantial delivery of PGN into the superficial layers (5 µm from the lumen) of the arteries with and without ultrasound, [(1951.17 relative fluorescence units (RFU) vs. 1901.17 RFU; P-value = 0.939)]. With ultrasound activation there was increased penetration of PGN into the deeper arterial layers (up to 35 µm from the lumen) [(13195.25 RFU vs. 7681.00 RFU; P-value = 0.005)]. These pre-clinical data demonstrate ultrasound mediated therapeutic vascular delivery to deeper layers of the injured arterial wall. This model has the potential to reduce peri- stent restenosis.

Atrial Thrombus and Embolic Stroke in a Patient With Surgical Appendage Ligation and Maze Procedure.

This case illustrates the incomplete protection of surgical ligation of left atrial appendage and maze procedure at the time of mitral valve replacement against thromboembolic complications and recurrence of atrial fibrillation. The utility of surgical left atrial appendage ligation as stroke prophylaxis and identification of selected high-risk subjects are reviewed. (Level of Difficulty: Intermediate.).

Stabilizing Peri-Stent Restenosis Using a Novel Therapeutic Carrier.

Late in-stent restenosis remains a significant problem. Bare-metal stents were implanted into peripheral arteries in miniature swine, followed by direct intra-arterial infusion of nitric oxide-loaded echogenic liposomes (ELIPs) and anti-intercellular adhesion molecule-1 conjugated ELIPs loaded with pioglitazone exposed to an endovascular catheter with an ultrasonic core. Ultrasound-facilitated delivery of ELIP formulations into stented peripheral arteries attenuated neointimal growth. Local atheroma-targeted, ultrasound-triggered delivery of nitric oxide and pioglitazone, an anti-inflammatory peroxisome proliferator-activated receptor-γ agonist, into stented arteries has the potential to stabilize stent-induced neointimal growth and obviate the need for long-term antiplatelet therapy.

A Critical Role of PCSK9 in Mediating IL-17-Producing T Cell Responses in Hyperlipidemia.

We previously demonstrated that atherogenic Ldlr -/- Apobec1 -/- (LDb) double knockout mice lacking both low-density lipoprotein receptor (LDLR) and apolipoprotein B mRNA-editing catalytic polypeptide-1 (Apobec1) had increased serum IL-17 levels, with T cell programming shifted towards Th17 cells. In this study, we assessed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in T cell programming and atherogenesis. We deleted the Pcsk9 gene from LDb mice to generate Ldlr -/- Apobec1 -/- Pcsk9 -/- (LTp) triple knockout mice. Atherosclerosis in the aortic sinus and aorta were quantitated. Lymphoid cells were analyzed by flow cytometry, ELISA and real-time PCR. Despite of dyslipidemia, LTp mice developed barely detectable atherosclerotic lesions. The IL-17, was very low in plasma and barely detectable in the aortic sinus in the LTp mice. In the spleen, the number of CD4+CD8- cells and splenocytes were much lower in the LDb mice than LTp mice, whereas, the IL-17-producing cells of γδTCR+ T cells and effector memory CD4+ T cells (CD44hiCD4+) in the spleen were significantly higher in the LDb mice than in the LTp mice. The Rorc mRNA expression levels were elevated in LDb mice compared to LTp mice. When re-stimulated with an anti-CD3 Ab, CD44hiCD4+ T cells from LDb mice secreted more IL-17 than those from LTp mice. T cells from LDb mice (with PCSK9) produce more IL-17 at basal and stimulated conditions when compared with LTp mice (without PCSK9). Despite the dyslipidemic profile and the lack of LDLR, atherogenesis is markedly reduced in LTp mice. These results suggest that PCSK9 is associated with changes in T cell programming that contributes to the development of atherosclerosis.

CT imaging of myocardial scar burden with CNA35-conjugated gold nanoparticles.

Management of patients suffering from myocardial infarction (MI) is based on the extent of coronary artery disease and myocardial scar burden. We have developed a potentially clinically-useful X-ray molecular imaging contrast agent based on gold nanoparticle (AuNPs) functionalized with collagen-binding adhesion protein 35 (CNA35) with the capabilities of achieving prolonged blood pool enhancement for vascular imaging of the coronary arteries and specific targeting of collagen within myocardial scar. At a concentration of ~ 45 mg Au/ml, AuNPs maintained a stable blood pool enhancement at 142-160 HU within an hour of intravenous administration. At 6 hours, specific signal enhancement was detected in the myocardium scar in rats injected with CNA35-AuNPs, but not with control AuNPs or in control animals. In conclusion, CNA35-AuNPs may be considered as a CT contrast agent for both vascular imaging of coronary artery disease and molecular imaging of myocardial scar in the heart.

Pushing the limits of immune-related response: a case of "extreme pseudoprogression".

The advent of immune checkpoint targeted immunotherapy has seen a spectrum of immune-related phenomena in both tumor responses and toxicities. We describe a case of pseudoprogression that pushes the limits of immune-related response criteria and challenges the boundaries and definitions set by trial protocols. A middle-aged man with conventional clear cell renal cell carcinoma (RCC) had received multiple prior systemic treatments including vascular endothelial growth factor receptor tyrosine kinase inhibitors, as well as multiple surgeries and radiotherapy treatments. He was eventually started on nivolumab-the anti-programmed death receptor-1 monoclonal antibody approved for the treatment of advanced RCC. Clinical deterioration was observed soon after a 100 mg dose of nivolumab, with onset of acute renal failure and declining performance status. Radiologic progression was documented in multiple sites including worsening tumor infiltration of his residual kidney. The patient was on palliative treatment and visited by the home hospice team in an end-of-life situation. The patient unexpectedly improved and went on to achieve a durable tumor response. The case is illustrative of an extreme manifestation of pseudoprogression, and impels us to probe the assumptions and controversies surrounding this phenomenon.

Loss of Smooth Muscle α-Actin Leads to NF-κB-Dependent Increased Sensitivity to Angiotensin II in Smooth Muscle Cells and Aortic Enlargement.

RATIONALE: Mutations in ACTA2, encoding the smooth muscle isoform of α-actin, cause thoracic aortic aneurysms, acute aortic dissections, and occlusive vascular diseases. OBJECTIVE: We sought to identify the mechanism by which loss of smooth muscle α-actin causes aortic disease. METHODS AND RESULTS: Acta2-/- mice have an increased number of elastic lamellae in the ascending aorta and progressive aortic root dilation as assessed by echocardiography that can be attenuated by treatment with losartan, an angiotensin II (AngII) type 1 receptor blocker. AngII levels are not increased in Acta2-/- aortas or kidneys. Aortic tissue and explanted smooth muscle cells from Acta2-/- aortas show increased production of reactive oxygen species and increased basal nuclear factor κB signaling, leading to an increase in the expression of the AngII receptor type I a and activation of signaling at 100-fold lower levels of AngII in the mutant compared with wild-type cells. Furthermore, disruption of smooth muscle α-actin filaments in wild-type smooth muscle cells by various mechanisms activates nuclear factor κB signaling and increases expression of AngII receptor type I a. CONCLUSIONS: These findings reveal that disruption of smooth muscle α-actin filaments in smooth muscle cells increases reactive oxygen species levels, activates nuclear factor κB signaling, and increases AngII receptor type I a expression, thus potentiating AngII signaling in vascular smooth muscle cells without an increase in the exogenous levels of AngII.

CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis.

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with (111)InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

Nitric Oxide-Enhanced Molecular Imaging of Atheroma using Vascular Cellular Adhesion Molecule 1-Targeted Echogenic Immunoliposomes.

The aim of this study was to determine whether pre-treatment with nitric oxide-loaded echogenic liposomes (NO-ELIP) plus ultrasound can improve highlighting by molecularly targeted (anti-vascular cell adhesion molecule 1 [VCAM-1]) ELIP of atheroma components. Atherosclerotic animals were treated with anti-VCAM-1-ELIP or immunoglobulin (IgG)-ELIP. Each group was selected at random to receive pre-treatment with standard ELIP plus ultrasound, NO-ELIP without ultrasound and NO-ELIP plus ultrasound. Intravascular ultrasound highlighting data for the same arterial segments were collected before and after treatment. Pre-treatment with NO-ELIP plus ultrasound resulted in a significant increase in acoustic enhancement by anti-VCAM-1-ELIP (21.3 ± 1.5% for gray-scale value, 53.9 ± 3.1% for radiofrequency data; p < 0.001 vs. IgG-ELIP, p < 0.05 vs. pre-treatment with standard ELIP plus ultrasound or NO-ELIP without ultrasound). NO-ELIP plus ultrasound can improve highlighting of atheroma by anti-VCAM-1 ELIP. This NO pre-treatment strategy may be useful in optimizing contrast agent delivery to the vascular wall for both diagnostic and therapeutic applications.

Noninvasive detection of macrophages in atheroma using a radiocontrast-loaded phosphatidylserine-containing liposomal contrast agent for computed tomography.

PURPOSE: Macrophage plays an important role in plaque destabilization in atherosclerosis. By harnessing the affinity of macrophages to certain phospholipid species, a liposomal contrast agent containing phosphatidylserine (PS) and X-ray computed tomographic (CT) contrast agent was prepared and evaluated for CT imaging of plaque-associated macrophages in rabbit models of atherosclerosis. PROCEDURES: Liposomes containing PS and iodixanol were evaluated for their physicochemical characteristics, in vitro macrophage uptake, in vivo blood pool clearance, and organ distribution. Plaque enhancement in the aorta was imaged with CT in two atherosclerotic rabbit models. RESULTS: In vitro macrophage uptake of PS liposomes increased with increasing amount of PS in the liposomes. Overall clearance of PS liposomes was more rapid than control liposomes. Smaller PS liposomes (d = 112 ± 4 nm) were more effective than control liposomes of similar size or larger control and PS liposomes (d = 172 ± 17 nm) in enhancing aortic plaques in both rabbit models. CONCLUSIONS: Proper liposomal surface modification and appropriate sizing are important determinant for CT-based molecular imaging of macrophages in atheroma.

Nitric oxide pretreatment enhances atheroma component highlighting in vivo with intercellular adhesion molecule-1-targeted echogenic liposomes.

We present an ultrasound technique for the detection of inflammatory changes in developing atheromas. We used contrast-enhanced ultrasound imaging with (i) microbubbles targeted to intercellular adhesion molecule-1 (ICAM-1), a molecule of adhesion involved in inflammatory processes in lesions of atheromas in New Zealand White rabbits, and (ii) pretreatment with nitric oxide-loaded microbubbles and ultrasound activation at the site of the endothelium to enhance the permeability of the arterial wall and the penetration of ICAM-1-targeted microbubbles. This procedure increases acoustic enhancement 1.2-fold. Pretreatment with nitric oxide-loaded echogenic liposomes and ultrasound activation can potentially facilitate the subsequent penetration of targeted echogenic liposomes into the arterial wall, thus allowing improved detection of inflammatory changes in developing atheromas.

Nitric oxide improves molecular imaging of inflammatory atheroma using targeted echogenic immunoliposomes.

OBJECTIVE: This study aimed to demonstrate whether pretreatment with nitric oxide (NO) loaded into echogenic immunoliposomes (ELIP) plus ultrasound, applied before injection of molecularly targeted ELIP can promote penetration of the targeted contrast agent and improve visualization of atheroma components. METHODS: ELIP were prepared using the pressurization-freeze method. Atherosclerosis was induced in Yucatan miniswine by balloon denudation and a hyperlipidemic diet. The animals were randomized to receive anti-intercellular adhesion molecule-1 (ICAM-1) ELIP or immunoglobulin (IgG)-ELIP, and were subdivided to receive pretreatment with standard ELIP plus ultrasound, NO-loaded ELIP, or NO-loaded ELIP plus ultrasound. Intravascular ultrasound (IVUS) data were collected before and after treatment. RESULTS: Pretreatment with standard ELIP plus ultrasound or NO-loaded ELIP without ultrasound resulted in 9.2 ± 0.7% and 9.2 ± 0.8% increase in mean gray scale values, respectively, compared to baseline (p < 0.001 vs. control). Pretreatment with NO-loaded ELIP plus ultrasound activation resulted in a further increase in highlighting with a change in mean gray scale value to 14.7 ± 1.0% compared to baseline (p < 0.001 vs. control). These differences were best appreciated when acoustic backscatter data values (RF signal) were used [22.7 ± 2.0% and 22.4 ± 2.2% increase in RF signals for pretreatment with standard ELIP plus ultrasound and NO-loaded ELIP without ultrasound respectively (p < 0.001 vs. control), and 40.0 ± 2.9% increase in RF signal for pretreatment with NO-loaded ELIP plus ultrasound (p < 0.001 vs. control)]. CONCLUSION: NO-loaded ELIP plus ultrasound activation can facilitate anti-ICAM-1 conjugated ELIP delivery to inflammatory components in the arterial wall. This NO pretreatment strategy has potential to improve targeted molecular imaging of atheroma for eventual true tailored and personalized management of cardiovascular diseases.

CT imaging of myocardial scars with collagen-targeting gold nanoparticles.

In the setting of myocardial ischemia, recovery of myocardial function by revascularization procedures depends on the extent of coronary disease and myocardial scar burden. Currently, computed tomographic (CT) imaging offers superior evaluation of coronary lesions but lacks the capability to measure the transmural extent of myocardial scar. Our work focuses on determining if collagen-targeting gold nanoparticles (AuNPs) can effectively target myocardial scar and provide adequate contrast for CT imaging. AuNPs were coated with a collagen-homing peptide, collagen adhesin (CNA35). Myocardial scar was created in mice by occlusion/reperfusion of the left anterior descending coronary artery. Thirty days later, un-gated CT imaging was performed. Over 6h, CNA35-AuNPs provided uniform and prolonged opacification of the vascular structures (100-130 HU). In mice with larger scar burden, focal contrast enhancement was detected in the myocardium, which was not apparent within that of control mice. Histological staining confirmed myocardial scar formation and accumulation of AuNPs. FROM THE CLINICAL EDITOR: This team of investigators presents a collagen-targeting gold nanoparticle-based approach that enables the imaging of myocardial scars via CT scans in a rodent model. This information would enable clinicians to judge the recovery potential of myocardium more accurately than the current CT-scan based approaches.

Ultrasound-enhanced thrombolytic effect of tissue plasminogen activator-loaded echogenic liposomes in an in vivo rabbit aorta thrombus model--brief report.

OBJECTIVE: Ultrasound enhances thrombolysis when combined with a thrombolytic and a contrast agent. This study aimed to evaluate the thrombolytic effect of our tissue plasminogen activator (tPA)-loaded echogenic liposomes (ELIP) in an in vivo clot model, with and without ultrasound treatment. METHODS AND RESULTS: The femoral arteries of New Zealand White rabbits (n=4 per group) were cannulated. The abdominal aortas were denuded, and thrombi were created using a solution of sodium ricinoleate plus thrombin. Rabbits were then randomly selected to receive tPA-loaded ELIP (200 µg of tPA/5 mg of lipid) or empty ELIP with or without pulsed (color) Doppler ultrasound (5.7 MHz) for 2 minutes. Thrombus was imaged and echogenicity analyzed before and after ELIP injection. Blood flow velocities were measured at baseline, after clot formation, and serially after treatment up to 15 minutes. tPA-loaded ELIP highlighted thrombus in the abdominal aorta more effectively than empty ELIP (P<0.05). Ultrasound enhanced the thrombolytic effect of tPA-loaded ELIP, resulting in earlier and more complete recanalization rates (P<0.001). CONCLUSION: This study demonstrates effective highlighting of clots and thrombolytic effect of tPA-loaded ELIP in an in vivo rabbit aorta clot model. Doppler ultrasound treatment enhances this thrombolytic effect, resulting in earlier and more complete recanalization rates.

In vivo therapeutic gas delivery for neuroprotection with echogenic liposomes.

BACKGROUND: Ischemia-related neurological injury is a primary cause of stroke disability. Studies have demonstrated that xenon (Xe) may have potential as an effective and nontoxic neuroprotectant. Xe delivery is, however, hampered by lack of suitable administration methods. We have developed a pressurization-freeze method to encapsulate Xe into echogenic liposomes (Xe-ELIP) and have modulated local gas release with transvascular ultrasound exposure. METHODS AND RESULTS: Fifteen microliters of Xe were encapsulated into each 1 mg of liposomes (70% Xe and 30% argon). Xe delivery from Xe-ELIP into cells and consequent neuroprotective effects were evaluated with oxygen/glucose-deprived and control neuronal cells in vitro. Xe-ELIP were administered into Sprague-Dawley rats intravenously or intra-arterially after right middle cerebral artery occlusion. One-megahertz low-amplitude (0.18 MPa) continuous wave ultrasound directed onto the internal carotid artery triggered Xe release from circulating Xe-ELIP. Effects of Xe delivery on ischemia-induced neurological injury and disability were evaluated. Xe-ELIP delivery to oxygen/glucose-deprived neuronal cells improved cell viability in vitro and resulted in a 48% infarct volume decrease in vivo. Intravenous Xe-ELIP administration in combination with the ultrasound directed onto the carotid artery enhanced local Xe release from circulating Xe-ELIP and demonstrated 75% infarct volume reduction. This was comparable to the effect after intra-arterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction. CONCLUSIONS: This novel methodology may provide a noninvasive strategy for ultrasound-enhanced local therapeutic gas delivery for cerebral ischemia-related injury while minimizing systemic side effects.

In vivo volumetric intravascular ultrasound visualization of early/inflammatory arterial atheroma using targeted echogenic immunoliposomes.

OBJECTIVES: This study aimed to demonstrate three-dimensional (3D) visualization of early/inflammatory arterial atheroma using intravascular ultrasound (IVUS) and targeted echogenic immunoliposomes (ELIP). IVUS can be used as a molecular imaging modality with the use of targeted contrast agents for atheroma detection. Three-dimensional reconstruction of 2-dimensional IVUS images may provide improved atheroma visualization. MATERIALS AND METHODS: Atheroma were induced in arteries of Yucatan miniswine (n = 5) by endothelial cell denudation followed by a 4-week high cholesterol diet. The contralateral arteries were left intact and served as controls. Anti-intercellular adhesion molecule-1 (ICAM-1) and generic gammaglobulin (IgG) conjugated ELIP were prepared. Arteries were imaged using IVUS before and after ELIP injection. Images were digitized, manually traced, segmented, and placed in tomographic sequence for 3D visualization. Atheroma brightness enhancement was compared and reported as mean gray scale values. Plaque volume was quantified both from IVUS and histologic images. RESULTS: Anti-ICAM-1 ELIP highlighting of the atheroma in all arterial segments was different compared with baseline (P < 0.05). There was no difference in the mean gray scale values with IgG-ELIP. Arterial 3D IVUS images allowed visualization of the entire plaque distribution. The highlighted plaque/atheroma volume with anti-ICAM-1 ELIP was greater than baseline (P < 0.01). CONCLUSION: This study demonstrates specific highlighting of early/inflammatory atheroma in vivo using anti-ICAM-1 ELIP. Three-dimensional IVUS reconstruction provides good visualization of plaque distribution in the arterial wall. This novel methodology may help to detect and diagnose pathophysiologic development of all stages of atheroma formation in vivo and quantitate plaque volume for serial and long-term atherosclerotic treatment studies.

Electronic state distributions of YBa2Cu3O(7-x) laser-ablated plumes.

A systematic study of the spectrally resolved atomic emission arising from the laser ablation of YBa(2)Cu(3)O(7-x) targets in the presence of an oxygen background reveals statistically distributed populations with characteristic temporally averaged electronic temperatures of 0.28-0.38 eV, 0.28-0.37 eV, and 0.40-0.48 eV for neutral barium, yttrium, and copper, respectively. The higher electronic temperatures of the lighter copper atoms cannot be explained by the effects of temporal averaging, as the time-of-flight spectra are similar for all three atomic species. The electronic temperatures decline slowly with distance from the target, with a characteristic length of 20 to 200 mean free paths. The plume kinetic energies determined from gated imagery recorded time-of-flight spectra are much higher, 3-250 eV, suggesting a small fraction of the kinetic energy is converted to electronic excitation during each collision at the shock contact front. A correlation between electronic temperatures and shock strength reduces the dimensionality of the dependence on pressure and target distance. The dependence of electronic temperature on deposition condition is weak, suggesting the electronic state distribution is a poor candidate as a monitor for process control during the manufacture of coated conductors.

Nitric oxide-loaded echogenic liposomes for nitric oxide delivery and inhibition of intimal hyperplasia.

OBJECTIVES: We sought to develop a new bioactive gas-delivery method by the use of echogenic liposomes (ELIP) as the gas carrier. BACKGROUND: Nitric oxide (NO) is a bioactive gas with potent therapeutic effects. The bioavailability of NO by systemic delivery is low with potential systemic effects. METHODS: Liposomes containing phospholipids and cholesterol were prepared by the use of a new method, freezing under pressure. The encapsulation and release profile of NO from NO-containing ELIP (NO-ELIP) or a mixture of NO/argon (NO/Ar-ELIP) was studied. The uptake of NO from NO-ELIP by cultured vascular smooth muscle cells (VSMCs) both in the absence and presence of hemoglobin was determined. The effect of NO-ELIP delivery to attenuate intimal hyperplasia in a balloon-injured artery was determined. RESULTS: Coencapsulation of NO with Ar enabled us to adjust the amount of encapsulated NO. A total of 10 microl of gas can be encapsulated into 1 mg of liposomes. The release profile of NO from NO-ELIP demonstrated an initial rapid release followed by a slower release during the course of 8 h. Sixty-eight percent of cells remained viable when incubated with 80 microg/ml of NO/Ar-ELIP for 4 h. The delivery agent of NO to VSMCs by the use of NO/Ar-ELIP was 7-fold greater than unencapsulated NO. We discovered that NO/Ar-ELIP remained an effective delivery agent of NO to VSMCs even in the presence of hemoglobin. Local NO-ELIP administration to balloon-injured carotid arteries attenuated the development of intimal hyperplasia and reduced arterial wall thickening by 41 +/- 9%. CONCLUSIONS: Liposomes can protect and deliver a bioactive gas to target tissues with the potential for both visualization of gas delivery and controlled therapeutic gas release.