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. 2024 Feb 20;13(4):e031417.
doi: 10.1161/JAHA.123.031417. Epub 2024 Feb 14.

AT1-AA Is Produced in Offspring in Response to Placental Ischemia and Is Lowered by B-Cell Depletion Without Compromising Overall Offspring Health

Nathan Campbell  1 , Evangeline Deer  1 , Dylan Solise  2 , Denise C Cornelius  1   3 , Ty Turner  1 , Lorena M Amaral  1 , Owen Herrock  1 , Ariel Jordan  1 , Shivani Shukla  1 , Tarek Ibrahim  1 , Babbette LaMarca  1   2
Affiliations

AT1-AA Is Produced in Offspring in Response to Placental Ischemia and Is Lowered by B-Cell Depletion Without Compromising Overall Offspring Health

Nathan Campbell et al. J Am Heart Assoc. .

Abstract

Background: Preeclampsia, new-onset hypertension during pregnancy alongside other organ dysfunction, is the leading cause of mortality for the mother and low birth weight for the baby. Low birth weight contributes to high risk of cardiovascular disorders later in life. Women with preeclampsia have activated B cells producing agonistic autoantibodies to AT1-AA (angiotensin II type I receptor). We hypothesize that rituximab, a B cell-depleting chemotherapeutic, will deplete maternal B cells in reduced uterine perfusion pressure (RUPP) rats without worsening the effect of placental ischemia on pup growth and survival.

Methods and results: To test this hypothesis, the RUPP procedure was performed, and rituximab was continuously infused via miniosmotic pump. Maternal blood and tissues were collected. A separate group of dams were allowed to deliver, pup weights were recorded, and at 4 months of age, tissues were collected from offspring. Immune cells were measured via flow cytometry, and AT1-AA was quantified using a contraction bioassay. Blood pressure increased in RUPP rats and was normalized with rituximab treatment. RUPP offspring also had increased circulating B cells, cytolytic natural killer cells, and increased circulating AT1-AA, which were normalized with maternal rituximab treatment. This is the first study to analyze the AT1-AA in RUPP offspring, which was normalized with rituximab.

Conclusions: Our findings indicate that perinatal rituximab lowers maternal mean arterial pressure in RUPP rats and improves birth weight, circulating AT1-AA, and circulating natural killer cells, indicating that rituximab improves adverse fetal outcomes in response to placental ischemia.

Keywords: autoantibodies; immunology; offspring; preeclampsia.

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Figures

Figure 1
Figure 1. Changes in blood pressure and circulating agonistic autoantibodies against AT1‐AA following rituximab treatment.
A, MAP is increased in RUPP (n=19) animals compared with NP (n=16) and is normalized in RUPP+rituximab (n=21) animals. B, Circulating AT1‐AA measured using a cardiomyocyte contraction bioassay is increased in RUPP serum (n=6) compared with NP serum (n=6) and is normalized in RUPP+rituximab serum (n=6). Data are expressed as mean±SEM. **P<0.01, ***P<0.001, ****P<0.0001. AT1‐AA indicates angiotensin II type 1 receptor; ΔBPM, change in beats per minute; MAP, mean arterial pressure; NP, normal pregnant; and RUPP, reduced uterine perfusion pressure.
Figure 2
Figure 2. Rituximab treatment does not impact pup weight, placental weight, or fetal survival in RUPP rats.
A, RUPP pups (n=19) were significantly smaller than NP pups (n=16), and RUPP+rituximab pups (n=21) were not changed. B, RUPP placentas (n=19) were significantly smaller than NP placentas (n=16), and RUPP+rituximab placentas (n=21) were unchanged. C, RUPP (n=19) and RUPP+rituximab (n=21) animals had a significantly lower percentage of the fetus' surviving pregnancy compared with NP (n=16) animals. Data are expressed as mean±SEM. *P<0.05, **P<0.01, ****P<0.0001. NP indicates normal pregnant; and RUPP, reduced uterine perfusion pressure.
Figure 3
Figure 3. B cells and cytolytic NK cells are reduced in rituximab‐treated RUPP rats.
A, Circulating B cells are increased in RUPP animals (n=13) compared with NP animals (n=7) and are reduced in RUPP+rituximab animals (n=11). B, Placental B cells are unchanged in NP (n=5), RUPP (n=9), and RUPP+ritxuimab (n=7) animals. C, Circulating total NK cells are increased in RUPP animals (n=11) compared with NP (n=7) and RUPP+rituximab (n=11) animals. D, Circulating cytolytic NK cells are increased in RUPP animals (n=11) compared with NP (n=7) and RUPP+rituximab (n=11) animals. E, Placental total NK cells are increased in RUPP animals (n=9) compared with NP (n=5) animals. F, Placental cytolytic NK cells are increased in RUPP animals (n=9) compared with NP (n=5) and RUPP+rituximab (n=9) animals. Data are expressed as mean±SEM. *P<0.05, **P<0.01 ****P<0.0001. ANK44 indicates anti‐NK cell antibody 44; ANK61, anti‐NK cell antibody 61; CD, cluster of differentiation; NK, natural killer; NP, normal pregnant; and RUPP, reduced uterine perfusion pressure.
Figure 4
Figure 4. Rituximab treatment improves birth weight in male RUPP offspring and does not affect growth.
A, RUPP male offspring litters have significantly decreased birth weight compared with RUPP+rituximab, sham, and sham+rituximab male offspring litters. B, At 16 weeks of age there is no significant difference in weight between groups of male offspring or groups of female offspring. Data are expressed as mean±SEM. *P<0.05, **P<0.01. R indicates rituximab; and RUPP indicates reduced uterine perfusion pressure.
Figure 5
Figure 5. Rituximab treatment in RUPP rats reduces offspring circulating B cells and activated NK cells.
A, Male offspring of rituximab treated RUPP and sham dams have significantly decreased circulating B cells. Female offspring of rituximab‐treated sham dams have significantly increased circulating B cells. B, There is no significant change in the circulating helper T‐cell population in offspring of RUPP or sham dams following rituximab treatment. C, There is no significant change in the circulating total NK‐cell population in offspring of RUPP or sham dams following rituximab treatment. D, Male and female offspring of rituximab‐treated RUPP dams have significantly reduced circulating activated NK cells compared with male and female RUPP offspring. Data are expressed as mean±SEM. *P<0.05, **P<0.01. NK indicates natural killer; R, rituximab; and RUPP, reduced uterine perfusion pressure.
Figure 6
Figure 6. RUPP offspring tend to have increased blood pressure and circulating agonistic autoantibodies to AT1‐AA.
A, There is no significant difference in blood pressure between male offspring groups or between female offspring groups. Female offspring overall had significantly lower blood pressure than male offspring. B, RUPP male offspring had significantly increased circulating AT1‐AA compared with NP, sham+rituximab, and RUPP+rituximab male offspring. Data are expressed as mean±SEM. *P<0.05, **P<0.01. AT1‐AA indicates angiotensin II type 1 receptor; ΔBPM, change in beats per minute; MAP, mean arterial pressure; R, rituximab; Ritux, rituximab; and RUPP, reduced uterine perfusion pressure.
Figure 7
Figure 7. Study overview.
In this study, pregnant Sprague‐Dawley rats underwent RUPP surgery and treatment with rituximab on GD14. Animals in the maternal study underwent carotid catheterization on GD18, and on GD19 blood pressure was measured and tissues were collected. Animals in the offspring study were allowed to give birth between GD21 and GD23, and birth weight was measured within 12 hours. Offspring were aged to 4 months, at which point 1 male and 1 female per litter underwent carotid cauterization, and the next day blood pressure was measured and tissues were collected. Rituximab treatment during pregnancy improved maternal blood pressure, reduced agonistic autoantibodies against AT1‐AA production, and reduced NK‐cell activation. Rituximab treatment during pregnancy also improved male offspring birth weight, reduced adult AT1‐AA production, and reduced adult NK‐cell activation. AT1‐AA indicates angiotensin II type 1 receptor; GD, gestational day; NK, natural killer; and RUPP, reduced uterine perfusion pressure.
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