Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection

1When Rouget first discovered pericytes in 1873, he observed that these cells might serve as contractile entities to regulate microcirculatory blood flow because of their structure and position on microvessels. However, this idea was later challenged based on findings that microcirculatory blood flow is largely regulated by upstream arterioles rich in α-smooth muscle actin (α-SMA), the contractile protein present in vascular smooth muscle cells. A number of studies in the past three decades reported that most capillary pericytes in the central nervous system contained little or no α-SMA, whereas α-SMA was detected in pericytes located on pre-capillary arterioles and post-capillary venules. In contrast, recent in vitro and in vivo functional studies have demonstrated that a subgroup of capillary pericytes are contractile in the brain and retina, and have the capacity to regulate the microcirculation by contracting and relaxing in response to physiological (i.e. neurovascular coupling) as well as pharmacological stimuli. Importantly, Hall et al. showed that capillaries covered by pericytes dilate before arte-rioles in response to neuronal stimulus in situ. Recently, an increase in astrocytic calcium was shown to mediate brain capillary changes in response to metabolic demand. Similarly, Biesecker et al. showed that calcium signaling in Müller cell endfeet was sufficient to evoke capillary dilation in the retina. Kisler et al. reported that transgenic mice with a decreased number of pericytes had deficient neurovascular coupling and reduced oxygen delivery to the brain, providing additional evidence for the importance of pericytes in blood flow regulation. Collectively, these experiments strongly suggest the presence of a bona fide contractile machinery in capillary pericytes. However, a recent paper dismisses the idea of pericytes being contractile by redefining α-SMA-expressing pericytes as smooth muscle cells. To address this paradox between functional and histological findings, we hypothesized that small amounts of α-SMA in capillary pericytes may be rapidly depolymerized during transcardial perfusion and fixation thus evading detection by immunolabeling. Consistent with this idea, α-SMA in pericytes has been detected by electron microscopy in which small amounts of α-SMA can be identified or by in vitro studies in which fixation is more rapidly achieved. Here, we show that when filamentous-actin (F-actin) depolymerization was prevented by F-actin stabilizing agents or by snap fixation, we detected α-SMA in a much larger fraction of microvascular pericytes, including capillary pericytes placed on the intermediate and deeper retinal vascular beds.

2To elucidate the current controversy on the presence of α-SMA in capillary pericytes and to test the hypothesis that low α-SMA immunoreactivity in pericytes could stem from a preparation artifact (Figure 1), we first examined F-actin protein expression in capillary pericytes using fluorescently-tagged phalloidin. Intriguingly, we found substantial F-actin in pericyte processes surrounding capillaries (Figure 1—figure supplement 1A–D). Encouraged by this observation, we tested several fixation methods to enhance α-SMA detection. Snap freeze fixation of retinas with methanol at −20°C, yielded almost twice as many α-SMA-immunopositive microvessels compared to paraformaldehyde (PFA) fixation (Methanol: 441 ± 28 vessels, PFA: 254 ± 63 vessels, p=0.023; ANOVA) (Supplementary file 1; Figures 1A–B, 2A, C–D and 3A–C). The number of α-SMA-labeled microvessels significantly increased in fourth order capillaries (Methanol: 144 ± 19 vessels, PFA: 60 ± 28 vessels, p=0.003; ANOVA) (Figures 1A–B, 2A, C–D and 3A–C). Ethanol fixation did not improve α-SMA immunostaining (Figure 1—figure supplement 2A–B), suggesting that the effect of methanol was likely due to a faster fixation and not the result of a nonspecific response (e.g. protein denaturation). No immunoreactivity was observed in negative controls when the anti-α-SMA primary antibody was omitted (Figure 2—figure supplement 1A–B). In addition to their typical bump-on-a-log appearance detected by lectin, the α-SMA-positive pericytes on capillaries were identified by NG2 or PDGRFβ co-immunolabeling (Figure 2B–C), or by their red fluorescence in NG2-DsRed mice, a transgenic line that allows selective visualization of pericytes (Figure 2D).

3To assess the potential contribution of γ-actin cross-reactivity to α-SMA immunolabeling, especially under fixation conditions that might perturb the equilibrium between different actin isoforms, we used a specific antibody against γ-actin after snap freeze fixation of retinas with methanol. Most of the γ-actin immunostaining ran longitudinally, parallel to the capillary and pericyte plasma membrane, unlike the circular α-SMA outlining the pericyte somata and processes around the capillaries (Figure 4A–G). We did not detect any γ-actin immunostaining in pericytes on capillaries after the 4th branch. We did not observe a redistribution of the immunostaining patterns of the two actin isoforms when F-actin depolymerization was inhibited with phalloidin (Figure 4D–G).

4Phalloidin or jasplakinolide binding to F-actin prevents depolymerization and fixes F-actin in the polymerized state, thus we reasoned that this strategy might enhance detection of α-SMA in retinal capillary pericytes. To test this, retinas were treated with phalloidin or jasplakinolide followed by fixation with methanol. We found that jasplakinolide or phalloidin treatment (Figures 1C, 2B, 3A–D and 5A–C) significantly increased the number of α-SMA-labeling on capillaries of 5th and 6th order compared to PFA fixation (5th order: jasplakinolide = 225 ± 28 vessels, phalloidin = 80 ± 19 vessels, PFA = 15 ± 7 vessels; 6th order: jasplakinolide = 123 ± 30 vessels, phalloidin = 23 ± 9 vessels, PFA = 0 ± 0 vessels, p=0.0001, ANOVA) (Figure 3A). Besides this treatment revealed additional α-SMA-immunolabeling on 7th order capillaries (Figures 2B and 5C) compared to methanol or PFA fixation (jasplakinolide: 31 ± 12 vessels, phalloidin: 20 ± 6 vessels, methanol: 0 ± 0 vessels; PFA: 0 ± 0 vessels, p=0.002; ANOVA) (Figure 3A). α-SMA-positive pericytes identified by methanol fixation or phalloidin treatment also expressed NG2 and their number was significantly higher compared to PFA-fixed retinas (PFA: 509 ± 30 vessels, methanol: 883 ± 56 vessels, phalloidin: 890 ± 138 vessels, p=0.035, ANOVA) (Figure 3B).

5Administration of phalloidin combined with methanol fixation also confirmed abundant α-SMA expression in capillary pericytes of the intermediate (PFA: 97 ± 23 pericytes, methanol: 424 ± 72 pericytes, phalloidin: 509 ± 79 pericytes, jasplakinolide: 497 ± 99 pericytes, p=0.03; ANOVA) and deeper (PFA: 4 ± 2.7 pericytes, methanol: 119 ± 27 pericytes, phalloidin: 260 ± 30 pericytes, jasplakinolide: 359 ± 96 pericytes, p=0.01; ANOVA) retinal plexus (Figures 2A–D and 3C–D, Figure 2—video 1). The vast majority of the α-SMA-positive capillary pericytes could only be visualized after methanol fixation at −20°C (Figures 2A, C–D and 3C–D; Figure 2—video 1) or upon in vivo administration of jasplakinolide or phalloidin (Figures 1C, 2B, 3A and C–D). Of interest, we found that pericytes at the junction of two capillaries (i.e. junctional pericytes) were more frequently α-SMA-positive and exhibited a characteristic circular staining pattern wrapping microvessels. In contrast, pericytes on the linear segment of the capillary, which displayed a helical strand-like staining pattern as reported by other groups, were less often α-SMA-positive.

6To further confirm α-SMA expression in pericytes, we sought to selectively reduce α-SMA expression using short interfering RNA (siRNA). α-SMA-siRNA significantly suppressed α-SMA expression in capillary pericytes 48 hr after intravitreal administration, while α-SMA expression in pericytes on upstream capillary branches and arterioles was less affected (Figure 6A–F). These results are in agreement with the idea that a small pool of α-SMA in capillary pericytes is quickly lost by depolymerization, hence making its histological detection difficult relative to α-SMA-rich pericytes on pre-capillary arterioles and vascular smooth muscle cells.

7In this report, we demonstrate that about 50% of NG2-positive pericytes on high order retinal capillaries (i.e. >5th) located in the intermediate and deeper retinal vascular plexus express α-SMA (Figure 3D). Previous reports relied on α-SMA immunohistochemistry involving the slow transcardial infusion of formaldehyde or PFA fixatives, thus lack of or weak α-SMA detection was likely due to rapid F-actin depolymerization leading to the disruption of the antibody-binding sites. Although antigen retrieval on PFA-fixed retinas revealed some α-SMA labeling in retinal microvessels, this signal was modest and only found in a few capillaries. Here, we show that faster tissue fixation with cold methanol strikingly increased the detection of α-SMA-positive pericytes. It is possible that the detection of the minute pool of α-SMA in small soma and processes of pericytes does not only depend on the fixation method, but might also be difficult to visualize in transgenic mice due to dispersion of the limited amount of membrane-bound reporter protein over the relatively large surface area of the pericyte membrane. Moreover, incomplete (mosaic-like) fluorescent protein expression after tamoxifen injection in inducible transgenic mice, in which Cre recombinase expression is driven by the α-SMA promoter, can also account for the failure to visualize low levels of α-SMA labeling at capillary pericytes. Importantly, we demonstrate that inhibition of α-SMA depolymerization in the intact eye using phalloidin or jasplakinolide, two F-actin stabilizing reagents with different pharmacological effects on F-actin, substantially increased visualization of α-SMA in retinal capillary pericytes, particularly those in the deeper retinal plexus. The ratio of α-SMA-positive pericytes was relatively lower to that found in upstream microvessels, consistent with previous reports showing less net O2 delivery from distal capillaries relative to proximal ones. Interestingly, knocking down α-SMA expression led to the disappearance of α-SMA immunostaining mainly in distal capillary pericytes, suggesting that the small pool of α-SMA in capillary pericytes is less stable than in vascular smooth muscle cells. Based on these findings, we conclude that a large population of pericytes, notably those at branching points of retinal microvessels, have the capacity to express α-SMA, which likely mediates their contraction. It has been suggested that γ-actin might contribute to pericyte contractility in cortical microvessels. However, our results do not support this hypothesis since we did not detect γ-actin-positive capillary pericytes on distal order branches after preventing F-actin depolymerization. The latter finding also indicates that the increase in α-SMA-positivity after phalloidin or jasplakinolide was not caused by cross-reactivity between the antibodies against γ- and α-isoforms or by accumulation of an excess amount of F-actin that might increase the immunostaining of all isoforms.

8Unlike peripheral tissues where the blood flow changes homogeneously, the density of pericytes is high in the CNS and retina, where the blood flow demand varies considerably between neighboring cell layers or groups. In vitro studies and recent in vivo brain studies have provided a growing body of evidence that capillary pericytes contract or dilate in response to vasoactive mediators. This blood flow regulation with fine spatial resolution may be essential for tissues with high functional specialization such as the brain and retina. The retinal capillary dilation in response to light stimulus is reportedly layer-specific. In line with our findings showing clear α-SMA expression in capillary pericytes of the intermediate plexus, the latter study reported robust capillary dilation in this region, but failed to detect α-SMA expression most likely due to the use of PFA-based fixation. A recent study using transgenic mice expressing fluorescent proteins driven by the NG2 or α-SMA promoters also confirmed the contractile capacity of microvascular pericytes in the cortex in vivo, however, it proposed a radical redefinition by classifying the NG2- and α-SMA-expressing (contractile) cells as smooth muscle cells, rather than pericytes, as they would have conventionally been named under the original Zimmermann definition used since 1923. The existence of at least three sub-classes of pericytes and transitional forms from smooth muscle cells has been a matter of confusion, emphasizing the need for an unambiguous definition of pericyte sub-classes and their corresponding specialized functions.

9In conclusion, we identify key components of the contractile machinery in a large population of pericytes in the healthy retina. The identification of α-SMA in capillary pericytes may contribute to clarify the current paradox between functional and histological studies, and expand our understanding of the mechanisms regulating blood flow at the single-capillary level in neurodegenerative conditions including stroke, retinal ischemia, diabetic retinopathy and Alzheimer’s disease.

10Seventy three Swiss albino (21–35 g), eleven NG2-DsRed mice were housed under diurnal lighting conditions (12 hr darkness and 12 hr light) and fed ad libitum. The number of animals used in each experiment is indicated in the corresponding legend and the Table 1, Supplementary file 1.

11Animal housing, care, and application of experimental procedures were all carried out in accordance with the institutional guidelines and approved by the Hacettepe University Animal Experiments Local Ethics Committee (2012/63), committee guidelines on animal resources at the University of Rochester (Rochester, New York), and the guidelines of the Canadian Council on Animal Care and the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM, Montreal, Quebec, Canada).

12Eyes were collected, fixed for 1 hr in 4% PFA at room temperature, and the retinas prepared as flattened whole-mounts by making four radial cuts. Whole retinas were labeled with lectin (20 μg/ml in PBS containing 0.5% Triton X-100 (PBST), Vector Laboratories, Burlingame, CA) or antibodies against neural glial antigen-2 (NG2) (Cspg4) (Millipore, Burllington, MA) and platelet-derived growth factor receptor beta (PDGRFβ) (Pdgrfb) (Abcam, Cambridge, UK). Secondary antibody was anti-rabbit IgG conjugated to Cy2 (Jackson ImmunoResearch, West Grove, PA). Briefly, retinas were permeabilized by freezing and thawing in PBST (−80°C for 15 min, room temperature for 15 min), washed 3 times for 10 min, and incubated in 2% PBST at 4°C overnight. The retinas were washed in PBST 3 times for 5 min, incubated in blocking solution (10% fetal bovine serum or normal goat serum in PBST) for 1 hr at room temperature, and then, incubated with each primary antibody diluted in blocking solution (5 μg/ml) at 4°C overnight. The following day, samples were washed in PBST 3 times for 5 min and incubated with secondary antibody diluted in blocking solution (3 μg/ml) for 4 hr at room temperature. We mounted retinas, vitreal side up, on slides and covered them with anti-fade reagent containing Hoechst-33258 to label cellular nuclei (Molecular Probes, Eugene, OR). Retinas were imaged under a light microscope (400x, Eclipse E600, Nikon Instruments Inc., Melville, NY) equipped with a manually controlled specimen stage for X, Y, and Z-axis, a color camera (model DXM1200, Nikon Instruments Inc.), a fluorescent light source (HB-10104AF, Nikon Instruments Inc.), and an image analysis software (NIS-Elements, Version 3.22, Nikon Instruments Inc.). Confocal images of the stained sections were obtained with a Zeiss LSM-510 confocal laser-scanning microscope equipped with a diode laser 488 nm and 561 nm source for fluorescence illumination, and a Leica TCS SP8 DLS (Leica, Wetzlar, Germany) confocal laser-scanning microscope, with a X-, Y-, and Z-movement controller, and a high-resolution PMT (Zeiss, Oberkochen, Germany) and HyD (Leica) detectors. Panoramic pictures of retina were generated by tiling individual images (20x). Samples were visualized with an Apotome fluorescent microscope (Apotome 2, Zeiss) that allowed optical sectioning and was equipped with an automatic controlled specimen stage for X, Y, and Z-axis, a color camera (Axiocam 509 mono, Zeiss), a fluorescent LED source (X-cite 120LEDmini, Excelitas, Waltham, MA), and an image analysis software (Zen, Zeiss) for image acquisition.

13After sacrificing the animals, eyes were fixed in PFA at room temperature or −20°C in 100%-methanol for 1 hr. Retinas were collected and permeabilized as described above. Tissue was blocked in 10% normal goat serum in PBST at room temperature. Since anti-α-SMA antibodies are commonly generated in mice to avoid non-specific binding to mouse epitopes of the tissue, first we incubated primary antibody against α-smooth muscle actin (α-SMA) (Acta2) (Sigma, San Louis, MO) separately with monofragments of the secondary antibody (Jackson Immunoresearch, West Grove, PA) for 2 hr (goat anti-mouse, for one retina: 2 µg of primary antibody with 1.5 µg of secondary antibody in 10 µl PBS). Then, we blocked the potential unbound monofragments by adding 200 µl of 10% normal mouse serum in PBS. Then, tissue was blocked (10% normal goat serum in PBS), and by incubation in the customized primary and secondary antibody mixture overnight at 4°C. Retinas were washed and mounted for visualization as described below.

14To prevent actin depolymerization, F-actin was fixed in vivo by 2 µl intravitreal injection of fluorescence phalloidin (200 U/ml, Biotium, Freemont, CA), non-fluorescent phalloidin oleate (5 µg/µl, Millipore, USA), or Jasplakinolide (10 µM, Abcam, UK). Two hrs later, animals were sacrificed and the eyes were collected and fixed in −20°C methanol for 1 hr. Retinas were harvested and subjected to the α-SMA immunostaining protocol described above. Under 200x magnification, we assigned an order number to each vessel segment before branching, starting from arterioles to capillaries, and the total number and the order of α-SMA-positive vessels was determined for each experimental condition. Under 400x magnification and for each fixation method, the number of α-SMA-positive microvessels in each retinal plexus and the total number of α-SMA+/NG2+ pericytes were quantified using a stereological approach. Thus, we analyzed an average of 140 disectors (field of view: 400 × 300 µm along Z-axis) per retina (same area between animal cohorts). The number of α-SMA-positive pericytes in each retinal plexus and the total number of α-SMA+/NG2+ pericytes were calculated using the fractionator equation as follows: total number of elements = Σ quantified elements/ssf X asf X tsf, where ssf is the section-sampling fraction (ssf = number of sections sampled/total sections), asf is the area-sampling fraction (asf = [a(frame)]/area x-y step between disectors), and tsf is the thick-sampling fraction (tsf = frame height/section thickness).

15A custom-designed, in vivo specific, HPLC purified α-SMA-siRNA (siRNA directed against Acta2) and a scrambled silencer select negative control siRNA were purchased (4457308 and 4404020, respectively, Ambion LifeTech, Carlsbad, CA). This α-SMA-siRNA was previously characterized in wound healing experiments in the murine liver. Each siRNA was injected into the vitreous using a 34-gauge Hamilton syringe (0.5 mg/ml siRNAs, total volume: 3 µl). Prior to injection, siRNAs were mixed with a transfection reagent to facilitate cell entry in vivo. Briefly, a transfection mixture composed of 3 µl In vivo-jetPEI (PolyPlus transfection, 201–10G, Illkirch-Graffenstaden, France) and 12.5 µl of 10% glucose in 9.5 µl of nuclease free water was added to the nucleic acid mixture (3.76 µl from 25 µg siRNA, 12.5 µl of 10% Glucose in 8.74 µl of nuclease free water), and incubated for 15 min at room temperature. Transfection mixture was prepared fresh before each knockdown experiment. Forty-eight hrs after intraocular siRNA delivery, mice were sacrificed. To check whether or not siRNAs were taken up by the cells, a fluorescent dye conjugated siRNA (BLOCK-iTTM Alexa Fluor 555 Fluorescent control; Thermo Fisher Scientific, Waltham, MA) was mixed with In vivo-jetPEI and delivered to retina as described above.

16Quantitative RT-PCR siRNA-treated retinas were removed precisely under sterile conditions to eliminate RNase contamination. The samples were stored in RNAlater solution (Qiagen, Hilden, Germany, 76104) at −20°C until RNA isolation. RNAs were extracted with RNeasy Mini Kit (Qiagen, 74104) according to instructions. Five hundred ng of total RNA for each sample was used in cDNA synthesis. cDNA synthesis was performed with random hexamer primers with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1621) according to instructions. cDNAs were stored at −20°C. Quantitative RT-PCR was performed with Taqman probe-based technology. Taqman gene expression master mix (ABI, Foster city, CA, 4369016), FAM-MGB labeled Taqman probes for mouse α-SMA gene (Assay ID: Mm00725412_s1) and mouse GAPDH gene (Assay ID: Mm9999991_g1) were used. PCR was carried out in triplicates in ABI OneStep Q RT-PCR machine (ABI). Thermal cyclic conditions were as follows: 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C 15 s, 60°C for 1 min. The relative expression values were calculated with ΔΔCt method. α-SMA expression in scrambled-siRNA delivered retinas (n = 3) was normalized to one fold expression and then α-SMA expression in α-SMA-siRNA delivered retinas (n = 3) was compared according to control siRNA group.

17Western blot analysis siRNA-treated retinas were removed and protein homogenates were isolated in the presence of a proteinase and phosphatase inhibitor cocktail containing RIPA buffer. Protein concentration was determined by Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225), and 40 μg protein per well was loaded to NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, NP0321BOX). Proteins were run and transferred to PVDF membranes, followed by incubation in blocking solution containing 5% BSA solution in TBS containing 0.5% tween-20 (TBST) overnight at 4°C. Blots were incubated with primary α-SMA antibody (2 µg/ml, Sigma, A2547-100UL) diluted in blocking solution for overnight at 4°C then with secondary goat anti-mouse HRP conjugated IgG (0.08 µg/ml, Santa Cruz Biotechnology, Dallas, TX, sc-516102) for 1 hr at room temperature. For loading control, blots were stripped with mild stripping buffer and blocked with 5% fat free milk powder solution in TBST for 1 hr at room temperature, incubated with primary β-tubulin III antibody (0.08 µg/ml, Sigma, USA, T2200) at 4°C for 20 min then with secondary goat anti-rabbit HRP conjugated IgG (0.05 µg/ml, Santa Cruz Biotechnology, sc-2357) for 30 min at room temperature. Bound antibodies were detected with SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher Scientific, 34095). Densitometric analyses were performed with ImageJ software.

18After extraction of whole-mount retinas, they were immunostained for α-SMA. Microvessels exhibiting continues α-SMA positivity in the superficial layer of whole mount retinas were counted based on capillary order. Counts were normalized compared to the α-SMA positive microvessel counts from scrambled-siRNA delivered retinas.

19All values are provided as the mean ±standard error of the mean (SEM). We evaluated all cohorts with normality (Shapiro-Wilk test) and variance (F-test) tests. For multiple comparisons of values of the stereological quantifications, we used Analysis of Variance (ANOVA) followed by Dunnett’s or Tukey’s test where appropriate. p≤0.05 was considered significant. For Western blot and qRT-PCR, two-tailed Student’s t-test was applied for statistical analysis. To analyze the α-SMA expression relative to capillary order, we used the specific non-parametric Jonckheere-terpstra test for trend analysis.