Investigating angiotensin II concentration in modulating Plasmodium falciparum infection and angiotensin receptor activation

Figures

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  Figure 1. Renin-angiotensin system (RAS) with its components. Angiotensinogen (AGT) and renin (REN) are released from liver and kidney, respectively. AGT is converted to angiotensin I by REN, and angiotensin I is converted to the main regulator peptide, angiotensin II by angiotensin-converting enzyme (ACE) and also to angiotensin (1-9) via angiotensin-converting enzyme2 (ACE2). Angiotensin (1-7) is generated from angiotensin II by ACE2 and angiotensin (1-9) by ACE. Further, Aminopeptidase A (APA) cleaves angiotensin II to angiotensin III that is subsequently cleaved to angiotensin IV via Alanyl aminopeptidase N (APN). Angiotensin II acts via its 2 cognate receptors, AT1R and AT2R. Angiotensin (1-7) counterbalances AT1R-mediated actions through Mas receptor and also via AT2R.

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  Figure 2. Invasion inhibition assay. Different concentration v/s percent parasitemia plotted in a bar graph chart where each bar represents mean ± standard deviation of control (negative and two positive) and 4 different concentrations (10–9 M, 10–8 M, 10–6 M, and 10–4 M) of angiotensin II. Overall statistical significance was estimated by one-way analysis of variance (ANOVA) with p-value of 0.0003 in GraphPad prism. Post hoc unpaired t-test revealed statistically significant difference between negative control and 10–9 M concentration (p<0.05; shown by *) and a highly statistically significant difference between negative control and 10–8 M and 10–6 M concentrations (p<0.01; shown by #). Pairwise p-values between negative control and different Ang II concentrations are mentioned above the respective bars. A representative image of Giemsa-stained smear in untreated versus Ang II treated (10–6 M) is also shown on the right. The infected RBCs are shown by arrow heads.

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  Figure 3. Human brain microvascular endothelial cell lines (HBEC5i) images showing the confluency of cells in hours after seeding (a) 7 hrs, (b) 24 hrs, (c) 36 hrs, and (d) 96 hrs as observed under phase contrast inverted microscopy at 400x magnification.

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  Figure 4. Calcium response to different doses of Ang II. The bars represent the response in the control (no angiotensin II), and at 3 different final concentrations (10–9 M, 10–8 M and 10–7 M) of angiotensin II. Data are expressed as mean ± SEM (Parameters: one-way analysis of variance (ANOVA) considering the p-value <0.05 as significant) in GraphPad prism.

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  Figure 5. cGMP response to different doses of Ang II. The bars represent the response in the control (no angiotensin II), and at 3 different final concentrations (10–9 M, 10–8 M and 10–7 M) of angiotensin II. Data are expressed as mean ± SEM of different concentrations of angiotensin II. (Parameters: one-way analysis of variance (ANOVA) considering the p-value <0.05 as significant) in GraphPad prism.

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  Figure 6. Multiple sequence alignment representation of native Ang II (wild; DRVYIHPF) and various modifications attempted in published literature. The native or unmodified octapeptide Ang II (DRVYIHPF) is depicted in the top and bottom rows of the alignment. Each of the 8 amino acid is separated by an asterisk to accommodate any downstream amino acid modification. The asterisk is also place before and after the first and last amino acid as well for the same. Amino Acid modifications are shown as coloured boxes with alphabets (amino acid substitutions), and special characters (for any other specified modification). Deleted amino acid loci are shown as black boxes. A (alanine), R (arginine), D (aspartic acid), C (cysteine), E (glutamic acid), G (glycine), H (histidine), I (isoleucine), K (lysine), F (phenylalanine), P (proline), Y (tyrosine), V (valine), $ (ornithine), # (D-ornithine), & (D-glutamic acid), @ (2,4-Di¬nitro-N-(2-phenyl¬ethyl) aniline). M1-M35 indicate the different type of modifications.