ALBERT

Description: One fundamental requirement shared by humans with all higher terrestrial life forms, including insect wings, higher land plants and other vertebrates, is a complex, fractally branching vascular system. NASA's VESsel GENeration Analysis (VESGEN) software maps and quantifies vascular trees, networks, and tree-network composites according to weighted physiological rules such as vessel connectivity, tapering and bifurcational branching. According to fluid dynamics, successful vascular transport requires a complex distributed system of highly regulated laminar flow. Microvascular branching rules within vertebrates, dicot leaves and the other organisms therefore display many similarities. One unifying perspective is that vascular patterning offers a useful readout that necessarily integrates complex molecular signaling pathways. VESGEN has elucidated changes in vascular pattern resulting from inflammatory, stress response, developmental and other signaling within numerous tissues and major model organisms studied for Space Biology. For a new VESGEN systems approach, we analyzed differential gene expression in leaves of Arabidopsis thaliana reported by GeneLab (GLDS-7) for spaceflight. Vascular-related changes in leaf gene expression were identified that can potentially be phenocopied by mutants in ground-based experiments. To link transcriptional, protein and other molecular change with phenotype, alterations in the Euclidean and dynamic dimensions (x,y,t) of vascular patterns for Arabidopsis leaves and other model species are being co-localized with signaling patterns of single molecular expression analyzed as information dimensions (i,j,k,...). Previously, Drosophila microarray data returned from space suggested significant changes in genes related to wing venation development that include EGF, Notch, Hedghog, Wingless and Dpp signaling. Phenotypes of increasingly abnormal ectopic wing venation in the (non-spaceflight) Drosophila wing generated by overexpression of a Notch antagonist were analyzed by VESGEN. Other VESGEN research applications include the mouse retina, GI and coronary vessels, avian placental analogs and translational studies in the astronaut retina related to health challenges for long-duration missions.

Description: Significant risks for visual impairment were discovered recently in astronauts following spaceflight, especially after long-duration missions.1 We hypothesize that microgravity-induced fluid shifts result in pathological changes within the retinal vasculature that precede visual and other ocular impairments. We therefore are analyzing retinal vessels in healthy subjects with NASA's VESsel GENeration Analysis (VESGEN) software2 before and after head-down tilt (HDT), a ground-based microgravity analog For our preliminary study of masked images, two groups of venous trees with and without small veins (G7) were clearly identified by VESGEN analysis. Upon completing all images and unmasking the subject status of pre- and post- HDT, we will determine whether differences in the presence or absence of small veins are important correlates, and perhaps reliable predictors, of other ocular and physiological adaptations to prolonged HDT and microgravity. Greater peripapillary retinal thickening was measured following 70-day HDT bed rest than 14-day HDT bed rest, suggesting that time of HDT may increase the amount of optic disc swelling.3 Spectralis OCT detected retinal nerve fiber layer thickening post HDT, without clinical signs of optic disc edema. Such changes may have resulted from HDT-induced cephalad fluid shifts. Clinical methods for examining adaptive microvascular remodeling in the retina to microgravity space flight are currently not established.

Description: Significant risks for visual impairment associated with increased intracranial pressure (VIIP) are incurred by microgravity spaceflight, especially long-duration missions. Impairments include decreased near visual acuity, posterior globe flattening, choroidal folds, optic disc edema and cotton wool spots. We hypothesize that microgravity-induced fluid shifts result in pathological changes within the retinal blood vessels that precede development of visual and other ocular impairments. Potential contributions of retinal vascular remodeling to VIIP etiology are therefore being investigated by NASAs innovative VESsel GENeration Analysis (VESGEN) software for two studies: (1) head-down tilt in human subjects before and after 70 days of bed rest, and (2) U.S. crew members before and after ISS missions. VESGEN analysis in previous research supported by the US National Institutes of Health identified surprising new opportunities to regenerate retinal vessels during early-stage, potentially reversible progression of the visually impairing and blinding disease, diabetic retinopathy.

Description: Significant risks for visual impairment were discovered recently in astronauts following spaceflight, especially after long-duration missions. We hypothesize that microgravity-induced fluid shifts result in pathological changes within the retinal vasculature that precede visual and other ocular impairments. We therefore are analyzing retinal vessels in healthy subjects before and after head-down tilt (HDT), a ground-based microgravity analog with NASA's VESsel GENeration Analysis (VESGEN) software. Methods. Spectralis infrared (IR) fundus images were collected from both eyes of 6 subjects before and after 70 days of bed rest at 6 degree HDT (NASA Campaign 11). For our retrospective study, branching patterns in arterial and venous trees are mapped by VESGEN into vessel branching generations (Gx) that are quantified by parameters such as densities of vessel length (Lv), area (Av), number (Nv) and fractal dimension (Df) as described previously for diabetic retinopathy (IOVS 51(1):498). Results are further assigned by VESGEN into groups of large (G1-3), medium (G4-6) and small (G7) vessels. Results. All subjects remained asymptomatic throughout duration of HDT. To date, we have analyzed one IR image from each of the 12 eyes. Interestingly, two groups of the masked study population identified by VESGEN are distinguished by the presence or absence of small veins (G7). For example, L7 and Av7 are 2.7+/-1.3 E-4 px/px2 and 7.2+/-3.6 E-4 px2/px2 in 6 retinas, but 0 in the other 6 retinas. Nonetheless, the space-filling properties of the entire venous trees were remarkably uniform by all parameters, such as Df = 1.56+/-0.02 for 6 retinas with G7 and 1.55+/-0.02 for retinas without G7. No small arteries (G7) were detected. Conclusions. For our preliminary masked analysis, two groups of venous trees with and without small veins (G7) were clearly revealed by VESGEN. Upon completing all images and unmasking the subject status of before and after HDT, we will determine whether differences in the presence or absence of small veins are important correlates, and perhaps reliable predictors, of other ocular and physiological adaptations to prolonged head-down tilt and microgravity. Clinical methods for examining adaptive microvascular remodeling in the retina to microgravity space flight are not currently established.

Description: Our hypothesis predicts that retinal blood vessels increase in density during early-stage progression to moderate nonproliferative diabetic retinopathy (NPDR). The renin-angiotensin system (RAS) is implicated in the pathogenesis of DR and in the function of circulating angiogenic cells (CACs), a critical bone marrow-derived population that is instrumental in vascular repair.

Description: Purpose: Our hypothesis predicts that blood vessels within the retina increase in density during early-stage nonproliferative diabetic retinopathy (NPDR), based on previous results of a small retrospective study. For the current prospective study, the remodeling of arteries and veins during progression of early NPDR is assessed by a repertoire of parameters that includes the fractal dimension (D(sub f) ). In complex structures such as branching vascular trees, D(sub f) is a sensitive measure of space-filing capacity. The renin-angiotensin system (RAS) is implicated in DR pathogenesis and the function of circulating angiogenic cells (CACs), a critical bone marrow-derived population instrumental in vascular repair. Methods: Arterial and venous branching patterns were extracted from images of 6 normal controls and 3 early NPDR subjects (2 moderate, 1 mild) acquired by Heidelberg Spectralis (Registered Trademark) OCT following fluorescein angiography (FA). The vascular branching patterns were analyzed by NASAs VESsel GENeration Analysis (VESGEN) software, in which skeletonized representations were generated automatically to yield D(sub f) by the box-counting method. For binary 2D images, D(sub f) varies between limiting Euclidean dimensions of 1 and 2. Peripheral blood of diabetics and controls was collected for CD34+ CAC isolation. The gene expression of RAS in CACs was assessed by qPCR for Mas receptor to Ang-(1-7). The vasoreparative function of the CACs was measured by migration ability toward CXCL12 (SDF-1). Results: By D(sub f), venous and arterial densities were 1.370 +/- 0.006 and 1.329 +/- 0.016 for early NPDR, compared to 1.318 +/- 0.012 and 1.320 +/- 0.036 for control. The space filling capacity in early NPDR measured by D(sub f), a sensitive parameter, therefore demonstrated a pronounced increase for veins, but not for arteries. Mas receptor mRNA in CACs was increased in diabetics without DR but reduced with onset of NPDR, indicating possible loss of compensation of protective RAS during early DR. Migratory dysfunction of CD34+ cells was further associated with DR. Conclusions: As assessed by the fractal dimension in our preliminary study, the space-filling capacity of veins, but not arteries, was greater in early NPDR than in control. Larger patient populations will be examined as we complete our ongoing longitudinal study. Results further suggest the protective RAS axis within diabetic CACs is lost early in DR and is associated with increased vascular remodeling as evidenced by VESGEN analysis.

Description: Significant risks for visual impairment associated with increased intracranial pressure (VIIP) are incurred by microgravity spaceflight, especially long-duration missions [1]. We hypothesize that microgravity-induced fluid shifts result in pathological changes within blood vessels of the retina that precede development of visual and other ocular impairments. Potential contributions of retinal vascular remodeling to VIIP etiology are therefore being investigated for two studies in 30deg infrared (IR) Heidelberg Spectralis(Registered Trademark) images with NASA's innovative VESsel GENeration Analysis (VESGEN) software [2,3]. The retrospective studies include: (1) before, during and after (pre, mid and post) 6 head-down tilt (HDT) in human subjects during 70 days of bed rest, and (2) before and after missions to the International Space Station (ISS) by U.S. crew members. Results for both studies are almost complete. A preliminary example for HDT is described below.

Description: Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice.

Description: Our hypothesis predicts that retinal blood vessels increase in density during early-stage progression to moderate nonproliferative diabetic retinopathy (NPDR). The prevailing paradigm of NPDR progression is that vessels drop out prior to abnormal, vision-impairing regrowth at late-stage proliferative diabetic retinopathy (DR). However, surprising results for our previous preliminary study 1 with NASA's VESsel GENeration Analysis (VESGEN) software showed that vessels proliferated considerably during moderate NPDR compared to drop out at both mild and severe NPDR. Validation of our hypothesis will support development of successful early-stage regenerative therapies such as vascular repair by circulating angiogenic cells (CACs). The renin-angiotensin system (RAS)is implicated in the pathogenesis of DR and in the function of CACs, a critical bone marrow-derived population that is instrumental in vascular repair.