Many circumstantial evidences from human and animal studies suggest that complement cascade dysregulation may play an important role in pregnancy associated complications including preeclampsia. Deletion of rodent specific complement inhibitor gene, Complement Receptor 1-related Gene/Protein y (Crry) produces embryonic lethal phenotype due to complement activation. It is not clear if decreased expression of Crry during pregnancy produces hypertensive phenotype. We downregulated Crry in placenta by injecting inducible lentivialshRNA vectors into uterine horn of pregnant C57BL/6 mice at the time of blastocyst hatching. Placenta specific downregulation of Crry without significant loss of embryos was achieved upon induction of shRNA using an optimal doxycycline dose at mid gestation. Crry downregulation resulted in placental complement deposition. Late-gestation measurements showed that fetal weights were reduced and blood pressure increased in pregnant mice upon downregulation of Crry suggesting a critical role for Crry in fetal growth and blood pressure regulation.
Complement cascade consisting of more than 30 proteins is a part of innate immune system. Complement is typically activated through three distinct pathways, the "classical", the "lectin" and the "alternative" pathways [[
Through complement activation or independently, components of the complement system such as C3, MBL and C1q play an important role in the success of normal pregnancy [[
Recently, the complement cascade has been implicated in the pathophysiology of preeclampsia. In preeclampsia patients, levels of complement activation by-products (C3a, Bb, C5a, and terminal complex MAC) are increased in circulation compared to normotensive pregnancies [[
Thus, many circumstantial evidence implicate the complement cascade in the pathophysiology of preeclampsia. However, it is not clear if there is a direct relationship between complement activation and preeclampsia pathophysiology, mainly hypertensive phenotype. An attractive approach to this end would be to induce complement activation by abrogating the expression of membrane bound complement regulators on placenta after implantation. Among the key membrane bound complement regulators in mice, expression of MCP was observed only in testis [[
Doxycycline inducible "SMARTvector Lentiviral" vectors enclosing Crry shRNA and non-target shRNA were purchased from Horizon discovery (Lafayette, CO, USA). Lentiviral particles enclosing these vectors were produced in our laboratory using kit as per the instructions provided by the manufacturer (Horizon discovery). Approval from institutional biosafety committee at Baylor College of Medicine was obtained for these procedures. The functional titer of lentiviral particles were determined by transducing into HEK293 cells before injecting into mice. Adjustments to the volume were made to account for the variations in transducing units between the batches of viral particles.
All animal procedures were approved by the Baylor College of Medicine institutional animal care and use committee and performed in accordance with NIH Guide for the Care and Use of Laboratory Animals. Eight week old C57BL/6J female mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were mated to C57Bl/6J proven breeder males for five days and the day of observed copulatory plug was identified as day post coitus (dpc) 0.5. On 3.5 dpc, a small incision was made on the right abdominal side of pregnant mice under isoflurane anesthesia to access right uterine horn. Crry shRNA or non-target shRNA lentiviral vectors (1250 transducing units/20 μl KSOM) were then injected into right uterine horn. Abdominal wall was closed using bio absorbable suture and wound clip was applied to close the skin. Meloxicam injection (ID) was given as analgesic. On 10.5 dpc doxycycline was added to drinking water to induce the shRNAs.
Tissue lysates containing 20μg protein were electrophoresed on 5–20% (w/v) polyacrylamide gradient gels (Thermo Fisher, Grand Island, NY, USA) and transferred on to Immune-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). Expression levels of Crry and C3 were probed using rabbit monoclonal antiCD46 antibody (Abcam, Cambridge MA, USA, cat# 108307). A secondary antibody goat anti-rabbit IgG-HRP (Southern Biotech, USA) and pierce ECL Western blotting substrate kit (Thermo Fisher) were used to develop the membranes. The Western bands were visualized using Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE, USA).
Immunofluorescence was performed on OCT-embedded frozen sections after fixing with ice-cold Methanol for 10 minutes at -20°C. The sections then were incubated with 3% Goat serum in PBS-Tween (0.01%) for 1 hour at room temperature in a humidified chamber to block nonspecific antibody binding. For co-staining, the primary antibodies, mouse anti-Crry antibody (Hycult Biotech, Wayne, PA, USA) and rabbit polyclonal anti-C3 (Abcam, Cambridge, MA, USA) were applied at 1:200 dilution overnight, followed by goat anti-mouse TX-red and anti-rabbit IgG Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature in a humidified chamber. Mouse and rabbit IgG (ready to use) (Dako, Glostrup, Denmark) was used as a negative control. Slides were then counterstained with Hoechst 33342 (Molecular Probes). Anti-fade mounting medium (Dako) was applied and slides were viewed and captured with a constant exposure time and aperture using a single threshold value under Olympus BX51 epifluorescence microscope, and images were recorded by a DP70 Digital Camera (Olympus Optical Co. Ltd., Tokyo, Japan). Subsequently, images were analyzed using ImageJ software and the numerical output of average intensity per nuclei was calculated.
On 17.5 dpc, BP (systolic, diastolic and mean) was measured by tail cuff method in non-anesthetized mice using new 8-channel CODA system (Kent Scientific Corporation, Torrington, CT, USA). During BP measurement mice were restrained in the nose cone holders with unrestricted breathing and clear visibility. To minimize restrain anxiety the mice were conditioned for 20 minutes daily for 3 days before measuring BP. An occlusion tail cuff and volume pressure recording (VPR) senor were gently inserted onto the tails of mice placed on a pre-warmed (37°C) platform. Recordings were performed in three sessions with 15 cycles in each session. The readings were averaged and averages of 3 sessions were again averaged to get the final reading.
After BP measurement, mice were euthanized and blood was collected to obtain serum. Placental and fetal weights were recorded. Placenta, kidney, liver, and uterine tissue were collected for Western blot, qPCR and immunofluorescence analysis.
For all the experiments data was presented as mean ± SEM. Differences between two groups were analyzed by student t test followed by Mann-Whitney whereas ordinary ANOVA was used for more than two groups. A p value of less than 0.05 was considered to be significant. GraphPad prism (GraphPad Software, San Diego, CA, USA) was used for statistical analysis.
We injected lentiviral particles enclosing either non-target (control group) or CrryshRNA vectors into right uterine horn of pregnant C57BL/6 mice (n = 5–7) around the time of blastocyst hatching (3.5 dpc). Since reduced expression of Crry before 9.5 dpc could affect embryo survival, we induced shRNA after 10.5 dpc. Initially, we titrated 3 doses of doxycycline (
Graph: Fig 1 Downregulation of placental Crry.Lentiviral vectors enclosing Crry or non-target shRNA were injected into right uterine horn of pregnant mice (n = 5–7) on 3.5 dpc, shRNA was induced on 10.5 dpc and placental expression of Crry on 17.5 dpc was analyzed. A) Representative Western blot showing downregulation of Crry in CrryshRNA mice. In each group placentas from both left and right uterine horn were used in the blot. B) Density analysis of Crry Western bands showing about 30% reduction in Crry levels in CrryshRNA mice compared to control mice (p<0.021). C) Representative immunofluorescence images showing reduced Crry expression in CrryshRNA mice. D) Semi quantitation of fluorescence intensity revealed significant downregulation (p = 0.04) of Crry in the labyrinthine zone of CrryshRNA mice compared to control mice. E) There was no change in fluorescence intensity in junctional zone between the two groups. F) In CrryshRNA mice downregulation of Crry on placentas from right uterine horn showed trend of increase compared to those from left uterine horn.
Western blot analysis further showed that in control mice there was no difference in the levels of Crry expression on placentas from left uterine horn compared to those from right uterine horn. In CrryshRNA mice, Crry expression levels showed a gradient. We observed a trend of increase in the magnitude of decrease in Crry levels on the placentas from right uterine horn compared to those from the left uterine horn in CrryshRNA mice (Fig 1F). However, the difference was not statistically significant.
Western blot analysis of Crry expression levels in uterus (Fig 2, left panel) and liver (Fig 2, right panel) showed that there was no difference between the two groups suggesting that Crry expression decreased in a placenta specific manner.
Graph: Fig 2 Decrease in Crry expression was placenta specific.Lentiviral vectors enclosing Crry or non-target shRNA were injected into right uterine horn of pregnant mice (n = 5–7) on 3.5 dpc, shRNA was induced on 10.5 dpc. Expression of Crry on 17.5 dpc in uterine tissue and liver was analyzed. Left panel is representative Western blot showing Crry levels in uterine tissue and density analysis. Right panel is representative Western blot showing Crry levels in liver and density analysis.
Overall, these results suggested that injecting vectors enclosing shRNA directly into uterine horn at the time of blastocyst hatching followed by shRNA induction starting at 10.5 dpc downregulated Crry expression levels on placentas. The Crry downregulation showed a gradient with more decreases in uterine horn that received vectors than in the contralateral horn. The decrease in the Crry expression was specific to labyrinthine zone. Further, decrease in the Crry expression appeared to be placenta specific since levels of Crry in the uterus and the liver were not different between CrryshRNA injected and control mice.
Western blot analysis revealed that at 17.5 dpc, deposition of C3b was significantly higher (p = 0.03) on placentas from CrryshRNA compared to those from control mice. About 30% more C3b was deposited on placentas from CrryshRNA mice than those from control mice (Fig 3, left panel). Immunofluorescence staining further confirmed that higher levels of C3b was deposited on placentas from CrryshRNA mice compared to those from control mice (Fig 3, right panel). Consistent with Crry expression levels, the immunofluorescence imaging also showed that the C3b deposition was significantly higher (p = 0.04) in labyrinthine zone of placentas from CrryshRNA mice Deposition of C3b in junctional zone was not different between the two groups. Taken together, the data showed that complement activation on placentas increased in CrryshRNA mice due to the downregulation of Crry.
Graph: Fig 3 Crry downregulation triggered complement deposition on placenta.Placental complement deposition on 17.5 dpc was measured after shRNA induction on 10.5 dpc in pregnant mice (n = 5–7). Left panel is representative Western blot showing placental C3b deposition levels and density analysis of placental C3b Western blots showing significantly (p = 0.03) higher deposition in CrryshRNA mice compared to control group. Right panel is representative immunofluorescence images showing increased C3b deposition in CrryshRNA mice and semi-quantitation of fluorescence intensity showing significantly higher C3b deposition (p = 0.04) in the labyrinthine zone of CrryshRNA mice compared to control mice. There was no change in fluorescence intensity in junctional zone between the two groups.
Since liver is the major source for circulating C3, we speculated that increased consumption of circulating C3 due to complement activation at placenta would stimulate C3 production in liver to replenish circulating levels. Therefore, we measured serum C3 levels and liver C3 mRNA and protein levels. RT-qPCR revealed that C3 transcript levels were significantly higher (p = 0.016) in the liver from CrryshRNA mice compared to control mice (Fig 4A). Western blot analysis revealed that liver C3 levels were not different between the CrryshRNA and control mice (Fig 4B and 4C). The ELISA results showed that serum C3 levels were not different between the two groups (Fig 4D). Taken together, these results suggested that liver C3 transcript levels were increased upon placental complement activation in CrryshRNA mice, and C3 protein levels were not higher corresponding to mRNA levels in CrryshRNA group indicating secretion of excess C3 into circulation. However, serum C3 levels were not higher in CrryshRNA group suggesting that there was a feed forward loop where C3 consumption at placenta depleted serum C3 levels resulting in upregulation in C3 production in liver and therefore, serum levels were replenished.
Graph: Fig 4 Feed-back upregulation of C3 in liver.Liver and serum C3 level on 17.5 dpc in pregnant mice (n = 5–7) were measured. A) RT-qPCR showing significantly (p = 0.012) elevated C3 transcript levels in liver from CrryshRNA mice compared to control mice. B) Representative Western blot showing C3 expression level in liver C) Density analysis of liver C3 Western bands reveled no difference in its levels between CrryshRNA and control groups D) Serum C3 levels measured by ELISA showing no difference between CrryshRNA and control mice.
The average litter size was not different between CrryshRNA and control mice (S2 Fig). The average fetal weight was significantly reduced (p = 0.048) in CrryshRNA mice (958.69±46.9 mg) compared to control group (783.69±18.02 mg) (Fig 5A). Placental weight was not significantly different (83.99±3.84 mg v/s 84.01±2.3 mg) between the two groups (Fig 5B). However, fetal-placental weight ratio was significantly decreased (p = 0.045) in Crry shRNA mice compared to control mice (Fig 5C) suggesting a reduced placental efficiency in CrryshRNA mice.
Graph: Fig 5 Decreased fetal weight in CrryshRNA mice.Fetal and placental weights were measured at 17.5 dpc soon after euthanasia (n = 5–7), A) Average fetal weight was significantly reduced (p = 0.048) in CrryshRNA mice compared to control group B) Average placental weight was not different between the two groups C) Fetal-placental weight ratio was significantly decreased (p = 0.045) in Crry shRNA mice compared to control mice.
Further, the systolic, diastolic and mean blood pressures were significantly higher (p = 0.026, p = 0.008 and p = 0.008 respectively) in CrryshRNA mice compared to control mice (Table 1). Overall these data indicated that placental complement activation due to the downregulation of Crry decreased fetal growth and increased maternal blood pressure.
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Table 1 Blood pressure elevated in CrryshRNA mice (n = 5–7).
Animal Group Systolic mmHg Diastolic mmHg Mean mmHg Non-target shRNA 133.95 ± 9.86 108.91 ± 7.2 118.08 ± 7.86 Crry shRNA 166.12 ± 4.08 135.07 ± 3.44 144.58 ± 3.36 0.026 0.008 0.008
We injected an inducible lentiviral vector enclosing shRNA targeted to Crry directly into right uterine horn in mice during the time of blastocyst hatching (3.5 dpc). The shRNA was induced starting at mid-gestation (10.5 dpc) using a dose of doxycycline that resulted in minimal loss of embryos. At late-gestation (17.5), we observed that Crry was downregulated in a placenta specific manner. With the optimal dose of doxycycline used Crry downregulation was about 30% compared to its expression levels in the control group. Further, the decrease in Crry was specific to labyrinthine zone. The downregulation of Crry resulted in increased complement activation on placentas from CrryshRNA mice. The C3 mRNA was upregulated in liver of CrryshRNA mice as a feed- forward response to decreased serum levels due to increased C3 turnover at fetal-maternal interface. Fetal weights were reduced and blood pressure increased in CrryshRNA mice suggesting a critical role for Crry in normal fetal growth and normal blood pressure. Since it is known that Crry plays an important role in the complement system homeostasis [[
Initially we attempted to employ published methods for placenta specific gene manipulation in rodents. Previously, several research groups used lentiviral vectors to manipulate genes in a placenta specific manner [[
In humans, functional analogues of rodent specific Crry, CD46 and CD55 play crucial role in the regulation of initial steps of complement cascade. Decreased expression of these proteins on placenta or loss of function mutations in them would increase complement activation causing elevated systemic C3a and increased cellular deposition of MAC. Several recent studies indicating elevated C3a, increased placental MAC deposition and association between CD46 mutations and preeclampsia suggest that in preeclampsia patients complement activation is increased at the feto-maternal interface possibly secondary to defective regulation [[
The magnitude of complement activation on placenta determined by the level of Crry expression may dictate the phenotype. In our mouse model we were able to alter the placental Crry levels using different doxycycline doses (S1 Fig). Placental Crry levels were reduced by about 70% with a high dose (500 μg/mL) of doxycycline resulting in increased embryo resorption. We observed about 30% Crry downregulation and a minimal embryo loss with the doxycycline dose of 75 μg/mL which was used in subsequent experiments. The difference in embryo survival can be attributed to the level of Crry since consistently equal lentiviral titers were used in these experiments. In addition, the doxycycline dose appear to dictate the location of Crry downregulation within placenta. With 75 μg/mL of doxycycline the Crry downregulation was observed only in the labyrinthine zone but not in the junctional zone. The cause of zonal specific effect of doxycycline may not be temporal or related to mode of administration since in mice, it has been shown that plasma concentrations of doxycycline increases rapidly and reaches plateau in 2 days when the doxycycline was given through drinking water [[
The amplitude of placental complement activation may determine the severity of preeclampsia. Preeclampsia is a heterogeneous disease with a range of manifestations from mild to severe. Furthermore, preeclampsia affects fetal growth in a manner related to the severity of the disease. Severe and early onset preeclampsia was found to be associated with a 12–23% reduction in birth weight [[
The data presented in this study suggested that increased complement activation related to the decreased placental complement regulatory protein play a role in the pathogenesis of preeclampsia -like symptoms, more akin to that in humans. Further characterization is underway to investigate if these mice also develop several other features observed in preeclampsia patients such as angiogenic imbalance, inflammatory biomarkers, glomerular endotheliosis and proteinuria, defective spiral artery remodeling, etc. The mechanisms that link complement mediated placental pathology to fetal growth and maternal blood pressure regulation will also need to be addressed in future studies. The data presented in this study clearly showed that placental Crry is critical for normal fetal growth and for maintaining normal blood pressure.
S1 Fig. Embryo survival upon induction of shRNA with different doses of doxycycline (n = 3).
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S2 Fig. Average litter size at 17.5 dpc in CrryshRNA and control mice.
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By Manu Banadakoppa; Kathleen Pennington; Meena Balakrishnan and Chandra Yallampalli
Reported by Author; Author; Author; Author