Federico Franchi

Noninvasive monitoring of oxidative stress in transplanted mesenchymal stromal cells

OBJECTIVES The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells. BACKGROUND In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function. METHODS Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions. RESULTS After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor. CONCLUSIONS Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

Evaluation of coronary adventitial vasa vasorum using 3D optical coherence tomography – Animal and human studies

Objectives This study sought to evaluate adventitial vasa vasorum (VV) in vivo with novel imaging technique of optical coherence tomography (OCT). Methods To verify OCT methods for quantification of VV, we first studied 2 swine carotid arteries in a model of focal angiogenesis by autologous blood injection, and compared microchannel volume (MCV) by OCT and VV by m-CT, and counts of those. In OCT images, adventitial MC was identified as signal-voiding areas which were located within 1 mm from the lumen-intima border. After manually tracing microchannel areas and the boundaries of lumen-intima and media-adventitial in all slices, we reconstructed 3D images. Moreover, we performed with OCT imaging in 8 recipients referred for evaluation of cardiac allograft vasculopathy at 1 year after heart transplantation. MCV and plaque volume (PV) were assessed with 3D images in each 10-mm-segment. Results In the animal study, among the 16 corresponding 1-mm-segments, there were significant correlations of count and volume between both the modalities (count r2=0.80, P<0.01; volume r2 =0.50, P<0.01) and a good agreement with a systemic bias toward underestimation with m-CT. In the human study, there was a significant positive correlation between MCV and PV (segment number=24, r2 =0.63, P<0.01). Conclusion Our results suggest that evaluation of MCV with 3D OCT imaging might be a novel method to estimate the amount of adventitial VV in vivo, and further has the potential to provide a pathophysiological insight into a role of the VV in allograft vasculopathy.

Polycystic kidneys have decreased vascular density: a micro-CT study.

Polycystic kidney disease (PKD) is a common cause of end‐stage renal failure and many of these patients suffer vascular dysfunction and hypertension. It remains unclear whether PKD is associated with abnormal microvascular structure. Thus, this study examined the renovascular structure in PKD.

Endothelial dysfunction occurs prior to clinical evidence of polycystic kidney disease.

Objective: Polycystic kidney disease (PKD), a monogenic disease with an autosomal dominant or an autosomal recessive form of inheritance (ARPKD), is the most common genetic cause of renal dysfunction and end-stage renal failure. In addition to the development of cysts, the autosomal form of PKD is associated with vascular endothelial dysfunction, a marker of vascular disease. Whether vascular endothelial dysfunction is also present in ARPKD, and its relationship with renal dysfunction remain to be determined. Methods: ARPKD rats (PCK model) and controls were studied at 6 and 10 weeks of age, and mean arterial pressure and renal function were measured. Aortic endothelial function was assessed using organ chamber techniques. Aortic endothelial cells (ECs) were isolated, characterized and their function studied. Results: Compared to controls, ARPKD animals had a decrease in the vasorelaxation to endothelium-dependent vasodilators, even prior to changes in mean arterial pressure or renal function. The abnormal vasoreactivity was corrected with L-arginine (a precursor of nitric oxide, NO), while the expression of endothelial NO synthase (eNOS) was unchanged. Furthermore, isolated ECs from 6-week-old ARPKD animals showed increased oxidative stress, with preserved eNOS expression and abnormal patterns of migration and angiogenic capacity (measured by the scratch and tube formation assays, respectively). Conclusion: ARPKD leads to impairments in aortic vascular function and ECs at an early stage, which can have significant functional consequences, potentially representing a novel therapeutic target in this disease.

Mesenchymal Stromal Cells Improve Renovascular Function in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is a common cause of end-stage renal failure, for which there is no accepted treatment. Progenitor and stem cells have been shown to restore renal function in a model of renovascular disease, a disease that shares many features with PKD. The objective of this study was to examine the potential of adult stem cells to restore renal structure and function in PKD. Bone marrow-derived mesenchymal stromal cells (MSCs, 2.5 × 105) were intrarenally infused in 6-week-old PCK rats. At 10 weeks of age, PCK rats had an increase in systolic blood pressure (SBP) versus controls (126.22 ± 2.74 vs. 116.45 ± 3.53 mmHg, p < 0.05) and decreased creatinine clearance (3.76 ± 0.31 vs. 6.10 ± 0.48 μl/min/g, p < 0.01), which were improved in PKD animals that received MSCs (SBP: 114.67 ± 1.34 mmHg, and creatinine clearance: 4.82 ± 0.24 μl/min/g, p = 0.001 and p = 0.003 vs. PKD, respectively). MSCs preserved vascular density and glomeruli diameter, measured using microcomputed tomography. PCK animals had increased urine osmolality (843.9 ± 54.95 vs. 605.6 ± 45.34 mOsm, p < 0.01 vs. control), which was improved after MSC infusion and not different from control (723.75 ± 56.6 mOsm, p = 0.13 vs. control). Furthermore, MSCs reduced fibrosis and preserved the expression of proangiogenic molecules, while cyst size and number were unaltered by MSCs. Delivery of exogenous MSCs improved vascular density and renal function in PCK animals, and the benefit was observed up to 4 weeks after a single infusion. Cell-based therapy constitutes a novel approach in PKD.

Non-invasive assessment of cardiac function in a mouse model of renovascular hypertension

Hypertension continues to be a significant cause of morbidity and mortality, underscoring the need to better understand its early effects on the myocardium. The aim of this study is to determine the feasibility of in vivo longitudinal assessment of cardiac function, particularly diastolic function, in a mouse model of renovascular hypertension. Renovascular hypertension (RVH) was induced in 129S1/SvImJ male mice (n=9). To assess left ventricular (LV) systolic and diastolic function, M-mode echocardiography, pulsed-wave Doppler echocardiography and tissue Doppler imaging were performed at baseline, 2 and 4 weeks after the induction of renal artery stenosis. Myocardial tissue was collected to assess cellular morphology, fibrosis, extracellular matrix remodeling and inflammation ex vivo. RVH led to a significant increase in systolic blood pressure after 2 and 4 weeks (baseline: 99.26±1.09 mm Hg; 2 weeks: 140.90±7.64 mm Hg; 4 weeks: 147.52±5.91 mm Hg, P<0.05), resulting in a significant decrease in LV end-diastolic volume, associated with a significant elevation in ejection fraction and preserved cardiac output. Furthermore, the animals developed an abnormal diastolic function profile, with a shortening in the E velocity deceleration time as well as increases in the E/e′ and the E/A ratio. The ex vivo analysis revealed a significant increase in myocyte size and deposition of extracellular matrix. Non-invasive high-resolution ultrasonography allowed assessment of the diastolic function profile in a small animal model of renovascular hypertension.

Scintillation sensing of 18 F-FDG for the detection of metabolically active atherosclerotic plaque

Atherosclerosis and its consequences are the main cause of mortality in the United States and the western world. Detection of metabolically active regions of atherosclerotic plaque can provide insights on those plaques that are prone to rupture, leading to a tissue infarction. The metabolic activity of these plaques can be identified using positron emission tomography (PET). However, such detection has been elusive in the coronary arteries due to motion and detection sensitivity. One of the techniques capable of improving precision as an affordable alternative is the use of an optical scintillating fiber for the detection of 18-Fluorine Fluorodeoxyglucose (18F-FDG), a compound that is accumulated in metabolically active areas of the atherosclerotic plaque. Here we report on a prototype scintillating fiber detection system designed to characterize the beta emission, particularly positron emission, by 18F-FDG. We demonstrate the ability to effectively measure the presence of 18F-FDG contained in-vivo along with the ability discriminate against ambient light based on energy. This paper presents a catheter-based hardware and software platform based on the use of a scintillating fiber optic catheter. The system measures a short (2–3 ns) burst of photons that are generated by the stimulation of the plastic scintillating fiber by a positron (633 kEv) emitted by the radioactive compound. It can be used to determine the presence of 18F-FDG in-vivo and accurately detect the active decay as the 18F-FDG retained in the metabolically active plaque. The proposed instrument was tested in-vitro and its sensitivity was also measured in-vivo. The developed system may be used in the detection of 18F-FDG, as surrogate of inflammation, providing critical in-vivo information of the metabolism in areas of inflammation.

Noninvasive Assessment of Cell Fate and Biology in Transplanted Mesenchymal Stem Cells

Recently, molecular imaging has become a conditio sine qua non for cell-based regenerative medicine. Developments in molecular imaging techniques, such as reporter gene technology, have increasingly enabled the noninvasive assessment of the fate and biology of cells after cardiovascular applications. In this context, bioluminescence imaging is the most commonly used imaging modality in small animal models of preclinical studies. Here, we present a detailed protocol of a reporter gene imaging approach for monitoring the viability and biology of Mesenchymal Stem Cells transplanted in a mouse model of myocardial ischemia reperfusion injury.

Regeneration ability of valvular interstitial cells from diseased heart valve leaflets

Regeneration of heart valves depends on the regeneration of cells residing in the heart valve leaflet, i.e., valvular interstitial cells (VICs). In this study, the regeneration capacity of VICs obtained from healthy and diseased valves of patients with various ages and genders were investigated. The cells obtained from the valves were cultured in vitro for 21 days on our developed standalone nanofibrous substrates. The substrates were pliable. Proliferation of cells, their morphologies, collagen deposition, and gene and protein expression were assayed to compare their regeneration capacity. VICs from healthy valves exhibited higher proliferation and cell spreading in comparison to VICs from diseased valves. However, collagen deposition by VICs from diseased valves was higher compared to the deposition by VICs from healthy valves, irrespective of age and gender of the patients. VICs on nanofibrous substrates showed high vimentin and collagen type I expression; expression of α-SMA showed the reverse trend demonstrating the importance of nanofibers in heart valve tissue engineering and regeneration. Although variations in proliferation, gene, and protein expression of VICs from healthy and diseased valves of patients with various ages and genders were observed, they were not statistically significant. Thus, VICs from a diseased valve maintain the potential to regenerate in a non-degenerative environment, providing an opportunity for tissue engineering of diseased valves.

Noninvasive Monitoring of the Mitochondrial Function in Mesenchymal Stromal Cells

PurposeMitochondria are a gatekeeper of cell survival and mitochondrial function can be used to monitor cell stress. Here we validate a pathway-specific reporter gene to noninvasively image the mitochondrial function of stem cells.ProceduresWe constructed a mitochondrial sensor with the firefly luciferase (Fluc) reporter gene driven by the NQO1 enzyme promoter. The sensor was introduced in stem cells and validated in vitro and in vivo, in a mouse model of myocardial ischemia/reperfusion (IR).ResultsThe sensor activity showed an inverse relationship with mitochondrial function (R2 = −0.975, p = 0.025) and showed specificity and sensitivity for mitochondrial dysfunction. In vivo, NQO1-Fluc activity was significantly higher in IR animals vs. controls, indicative of mitochondrial dysfunction, and was corroborated by ex vivo luminometry.ConclusionsReporter gene imaging allows assessment of the biology of transplanted mesenchymal stromal cells (MSCs), providing important information that can be used to improve the phenotype and survival of transplanted stem cells.