The study was performed in the Department of Periodontology of the Faculty of Dental Sciences, Sri Ramachandra University, Chennai, India. Ethical approval was obtained from the Institutional ethical committee of Sri Ramachandra University prior to the start of the study. Informed consent was obtained from all the study participants prior to recruiting them into the study. The study comprised of 3 groups viz. Group 1: healthy individuals (n=24), Group 2: patients on Nifedipine manifesting DIGO (n=18) and Group 3: patients on Nifedipine with no DIGO (non responders) (n=8). The inclusion criteria for group 1 were the presence of clinically healthy gingiva with absence of attachment loss and bleeding on probing and also the absence of any systemic diseases. The inclusion criteria for group 2 were hypertensive patients on Nifedipine 5mg once daily dose taken atleast for a minimum of 3 months with no other systemic disease who manifested the clinical signs of gingival overgrowth as described by Angelopolous and Goaz [14]. The gingival overgrowth severity index given by Pernu HE et al., [15] was used in the study based on which patients were assessed and gingival tissue samples were obtained from sites with an overgrowth severity score of 2 to achieve uniformity in sampling. The inclusion criteria for Group 3 were hypertensive patients on Nifedipine 5mg once daily dose taken atleast for a minimum of 3 months with no other systemic disease who did not manifest the clinical signs of gingival overgrowth. The exclusion criteria for the study were pregnancy, lactation, antibiotic and NSAID use in the last 6 months and intake of any other drugs that cause gingival overgrowth.

Medical fitness was obtained from the physician of the participants prior to collecting the gingival tissue samples. All necessary precautions were taken as hypertensive patients were recruited in the study. Plain lignocaine 2% without adrenaline was used as the local anaesthetic agent for the surgery. Gingival tissue samples were collected from healthy individuals and non responders during surgical crown lengthening done for prosthodontic purpose. Gingival tissue samples were obtained from patients on Nifedipine with DIGO during surgical gingivectomy. All participants underwent professional scaling and plaque control measures prior to the surgery to prepare the tissues and avoid excessive bleeding during surgery and to eliminate the confounding effects of dental plaque induced inflammation on Angiotensin levels. The gingival tissue samples were rinsed in saline after collection, homogenised by mechanical maceration in phosphate buffered saline and centrifuged at 10000 g for 15 minutes. The supernatant obtained was stored in -80 degrees Celsius till further processing. A commercially available ELISA kit (Cayman chemicals Inc) was used to estimate Angiotensin II in the samples. After sample purification, ELISA procedure was performed according to the instructions provided in the kit and the results were finally obtained by subjecting the 96 well microtitre plate to the ELISA reader at 414 nm. The results were compared with standards and blank wells provided in the kit.

The results were statistically analysed using SPSS software version 16. Multivariate analysis was performed using Kruskal Wallis test. To find the significance difference between the bivariate samples in independent groups the Mann-Whitney U test was used. In both the above statistical tools the probability value <0.05 is considered as significant and probability value of <0.01 is considered highly significant

The study comprised of 3 groups viz. Group 1: healthy individuals (n=24), Group 2 :patients on Nifedipine manifesting DIGO (n=18) and Group 3 : patients on Nifedipine with no DIGO (non responders) (n=8). The demographic details of the study participants are depicted in [Table/Fig-1].

The mean Angiotensin II, levels in the gingival homogenate samples of the study groups in depicted in [Table/Fig-2].

Multivariate analysis by Kruskal Wallis test revealed a statistically significant difference in Angiotensin II levels between the 3 groups with a p-value of .0001 denoting high statistical significance [Table/Fig-3].

Bivariate analysis by Mann Whitney U-test revealed statistically significant difference in angiotensin II values between the group pairs namely group 1 and group 2, group 2 and group 3, group 3 and group 1 with p-values of .001 denoting high statistical significance. The Angiotensin II values were highest in Group 2 [Table/Fig-4].

Though a relationship between Angiotensin II and Gingival overgrowth has been demonstrated in animals, to our knowledge this may be the first study to show a correlation between the two, in humans in vivo. Angiotensin II, the effector peptide of the Renin Angiotensin system is generated by cleavage of Angiotensinogen to Angiotensin I and finally to Angiotensin II. Angiotensin converting enzyme is responsible for the conversion mentioned above. However, enzymes such as chymase which is a product of mast cells could also mediate conversion of Angiotensinogen to Angiotensin I and Angiotensin II. Angiotensin II is regarded a proinflammatory, procoagulant and profibrotic cytokine and is known to exert its effects on target cells through 2 receptors AT1 and AT2.

The significant increase in Angiotensin II levels in the Nifedipine induced gingival overgrowth patients compared to healthy individuals and non responders as shown by our study, confirms the role of Renin Angiotensin system in DIGO. A study by Santos and co-workers has shown a fully operating RAS in rat gingiva [11] and recently we have shown the presence of Angiotensin Converting Enzyme in gingival fibroblasts (unpublished data). This fact may rule out the need of mast cell chymase in the gingiva for Angiotensin II generation. Evidence by the studies of Ohuchi and co-workers show the presence of AT1 and AT2 receptors for Angiotensin II in rabbit gingival fibroblasts [12]. In our lab too, we have shown the presence of AT1 and AT2 receptors in cultured human gingival fibroblasts (unpublished data). The presence of high amounts of Angiotensin II in the DIGO samples as shown in our study could lead to receptor mediated effects.

Angiotensin II was previously thought only as a vasoactive agent. But recent evidence suggests its role in fibrosis. A study by Kim and Iwao has reported the role of angiotensin II in myocardial fibrosis [16]. It has also been demonstrated that angiotensin II modulates renal cell growth and extracellular matrix synthesis by upregulating two significant growth factors namely Transforming Growth Factor β and Connective Tissue Growth Factor [10].

Inflammation of the gingiva induced by dental plaque has been regarded a pre requisite for drug induced gingival overgrowth. Angiotensin II levels could be modulated by inflammation. However in our study, we eliminated the confounding effects of plaque induced inflammation by performing scaling and plaque control measures and re-evaluating the patients gingival status prior to gingival tissue sample collection. Our findings are also supported by the fact that gingival fibroblasts in cultures where the inflammatory millieu cannot be created also respond to the drug administration and have shown molecular changes pertaining to fibrosis. The most probable reason could be the presence of Angiotensin II in the serum added to cultures. The significantly low levels of Angiotensin II in non responders (p<.01) suggests that along with the drug intake a high level of Angiotensin II is required to modulate the pathogenesis of DIGO.

The limitation of our study is the fact that we have not assessed the AT1 and AT2 receptor levels in the tissue samples. We are planning future in vivo and in vitro studies to validate the presence of a fully functional Renin Angiotensin system in the human gingiva.

In conclusion our study has shown increased levels of Angiotensin II in Nifedipine induced gingival overgrowth. Angiotensin II could modulate the pathogenesis of DIGO similar to renal and myocardial fibrosis. These effects could be mediated through the AT1 and AT2 receptors. If patients with hypertension could be pharmacologically managed with Angiotensin II antagonists like Losartan and Angiotensin receptor blockers, the occurrence of DIGO could be prevented. Clinical studies in this regard could benefit the human populations world wide.