Materials and Methods:
Vector construction and characterization
A secreted form of human ACE2 gene lacking the membrane domain, which has been previously
characterized1, was cloned into an AAV vector under the control of the CBA promoter and
packaged into serotype 2 viral particles. To construct the Ang-(1-7) expression vector, the coding
region for Ang-(1-7) peptide was cloned as a fusion protein with GFP reporter gene, separated by
a furin cleavage site (FC). The coding region for the fusion protein also contained secretion
signal. The control vector contained a secreted GFP coding region without peptide (sGFP). The
expression of the fusion sGFP-FC-Ang-(1-7) under the control of the CBA promoter in the AAV
vector was confirmed by tranfecting the HEK293 cells using this plasmid DNA . To ensure that
the fusion protein was indeed secreted, proteins isolated from the culture supernatants as well as
cell lysates from transfected, sham-transfected or untransfected cells were analysized by western
blotting. The Ang-(1-7) peptide was analyzed by Mass Spectrometry in the University of
Florida’s Mass Spectrometry Core Facility. Retinal protein extracts were also used to quantitate
Ang-(1-7) peptide using an EIA kit from Bachem (Bachem, San Carlos, CA) following the
instructions provided by the manufacturer.
Plasmids and AAV production
Recombinant viruses were produced and purified as previously described2, 3. Briefly,
HEK 293 cells were cotransfected with the appropriate construct DNA described above and the
helper plasmid pDG DNAs for 48–60 h. Cells were harvested, and the crude lysate purified
through an iodixanol step gradient followed by Mono-Q FPLC chromatography. The vector
genome (vg) titers of AAV2 particles were determined by real-time PCR.
Ocular injections
For ocular injections, eyes were dilated by topical administration of 1% atrophine sulfate
ophthalmic solution (Bausch & Lomb, FL) and 2.5% phenylephrine hydrochloride ophthalmic
solution (Akorn, IL). Animals were then anesthetized by ketamine (72mg/kg) /xylazine (4mg/kg)
intraperitoneal injection. Intravitreal injection was made through sclera/choroids and retina into
vitreous cavity using a 33-gauge beveled sharp needle (Hamilton Company, Nevada). One
microliter of AAV vector (~109 vector genome) was injected for each mouse eye and 2l was
injected in each rat eye. The control eye was either un-injected, sham injected with PBS, or an
AAV vector expressing the secreted GFP without ACE2 or Ang-(1-7).
Transgene expression from AAV vectors is usually detectable in the retina by two weeks after
ocular delivery of a conventional AAV2 vector, and reaches steady state levels around 4 weeks
following ocular delivery. To ensure sufficient therapeutic expression of both Ang-(1-7) and
ACE2 before the onset of diabetes, these AAV vectors were injected intravitreously two weeks
prior to STZ treatment to induce diabetes.
Retinal vascular permeability assay
Retinal vascular permeability was evaluated by albumin extravasation. Anesthetized
mice received intravenous injection of FITC-labeled albumin (100mg/Kg body weight, Sigma,
St.
Louis, MO). After 30 minutes animals were sacrificed, eyes enucleated, fixed in 4%
paraformaldehyde (freshly made in PBS, pH 7.4), and embedded in OCT. Frozen sections
(12um) were cut and mounted on slides. Extravasation of FITC-albumin from retinal vessels
was evaluated in serial cross-sections (10 sections with 50um interval, representing total 500um
thickness) by fluorescence imaging and quantified from sections by measuring the fluorescent
intensity using a Zeiss AxionVision software system.
Trypsin digest preparation of retinal vasculature
Retinal vasculature was prepared using the method described by Kuwabara and Cogan4 with
minor modifications. Briefly, eyes were fixed in 4% paraformaldehyde freshly made in PBS
overnight after enucleation. Retinas were dissected out from the eyecups, washed in water
overnight, and then digested in 3% trypsin (GIBCO-BRL) for 2-3 hr at 37C. The tissue was then
transferred into water and the network of vessels was freed from adherent retinal tissue by
gentle shaking and manipulation under a dissection microscope. The vessels were then
mounted on a clean slide, allowed to dry, then stained with PAS-H&E (Periodic Acid SolutionHematoxylin, Gill No.3, Sigma, St. Louis, MO). After staining and washing in water, the tissue
was then dehydrated and mounted in Permount mounting media. The prepared retinal vessels
were imaged using Zeiss microscope equipped with a high resolution digital camera (AxioCam
MRC5, Zeiss Axionvert 200) using both 20X and 40X objective lenses. 6-8 representative,
nonoverlapping fields from each quadrant of the retina were imaged. Acellular capillaries are
counted from images for each retina, and expressed as number of acellular vessels per mm2.
Retinal capillary basement membrane evaluation
The basement membrane thickness of retinal capillaries was evaluated by transmission
electron microscopy (TEM). Anesthetized mice were perfused with fixative containing 2%
paraformaldehyde and 2% glutaraldehyde, eyes enucleated and immersed in the same fixative
overnight after removing the cornea and lens. The eyes were then post-fixed in 2% OsO4,
dehydrated in ethanol series, embedded in epoxy resin. Thin sections (0.5um) were stained with
toluidine blue for orientation and identification of capillaries. Ultrathin sections (60nm) were
counterstained with uranyl acetate and lead citrate and examined by TEM. Retinal capillary
basement membrane thickness (CBMT) was measured from TEM images captured from deep
capillaries residing between OPL and INL. Minimally 10 capillaries from central, mid-, and
peripheral retinas were measured for each eye, at least 30 measurements were taken per
capillary.
Immunocytochemistry
Enucleated eyes were fixed in 4% paraformaldehyde freshly made in phosphate
buffered saline (PBS) at 4ºC overnight. Eyecups were cryoprotected in 30% sucrose/ PBS for
several hours or overnight prior to quick freezing in optical cutting temperature (OCT)
compound. Then 12um thick sections were cut at –20 to –220C. FITC- conjugated monoclonal
antibodies against mouse CD11b and CD45 were purchased from BD BioSciences (San Jose,
CA). Sections were pre-incubated with 5% BSA for 10 min, followed by incubation with
primary antibodies (1:100 in 1% BSA) overnight at 4ºC. An alkaline phosphotase- conjugated
anti-FITC antibody (Sigma) was used as the secondary antibody, and signal was detected using
NBT/BCIP as substrate (Roche, IN). Levamisole (Vector Laboratory, CA) was added to the
sections to remove the endogenous phosphatase activity. Positive cells were counted from at
least 12 sections at 100um intervals for each eye.
ACE and ACE2 activities
ACE and ACE2 activity were determined using assays based on internally quenched fluorescent
substrates (Abz-Phe-Arg-Lys[Dnp]-Pro-OH, M-2590 for ACE and Abz-Ser-Pro-3-nitro-Tyr-OH,
M-2660 for ACE2, both from Bachem, Torrance, CA) as described by Alves et al5 for ACE and
Yan et al6 for ACE2.
Thiobarbituric acid reactive substances (TBARS) Assay
Oxidative damage was assessed by the use of the thiobarbituric acid-reactive substances
(TBARs) assay, which detects any thiobarbituric acid-reactive substance such as
malondialdehyde7, in retinal homogenates using method modified from Dawn-Linsley et al8.
Briefly, retinal homogenate (10 g total protein for each retina) in 4001 of 1M copper
sulfate/5mM HEPES was mixed with 1 ml of a 0.375% TBA/15% trichloroacetic acid in 0.25N
HCl, incubated for 30 min at 90 ◦C, and clarified by centrifugation (1500 rpm for 10 min). The
resulting supernatant was aspirated and fluorescence quantified in a fluorescent
spectrophotometer (excitation 520 nm, emission 553 nm) by comparison with a standard curve of
tetramethoxypropane in HCl. Each sample was run in duplicates, and data were averaged from 68 retinas for each group.
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