MATERIALS AND METHODS

Experimental materials: Cadmium salt [Cadmium chloride (CdCl₂)] was used in present study to find out its effects on the process of angiogenesis using the chicken chorioallantoic membranes (CAM) model^14,15.

Sample size: The embryonated chicken eggs (n = 20, 61±3 g of 9-11 days of age) were procured from State Poultry Farm, Raisen Road, Bhopal, Madhya Pradesh.

Experimental groups: The eggs were divided into two experiments groups (10 eggs in each), comprising of one control group (Group I) and one treatment groups (Group II, treated with CdCl₂ salt).

Procedure: The egg surfaces were cleaned and wiped with 70% ethanol. Two hundred microliters of 0.5 M of CdCl₂ was directly introduced onto the CAM of respective group of embryonated eggs using 26 gauge hypodermic syringe along with antibiotics (Ampicillin 50 μg mL^–1, streptomycin 10 μg mL^–1) and antimycotic solution (Amphotericin 10 μg mL^–1). Phosphate Buffered Saline (PBS) with antibiotic and antimycotic solution was given to the control group. The eggs were resealed with cellophane tape and incubated for 72 h in a humid incubator chamber at 37±1°C.

Observation methods: The eggs were then opened to observe the effects of CdCl₂ on vascularisation. The CAM was grossly examined, harvested and subjected to histopathological analysis using hematoxylin and eosin stain.

RESULTS AND DISCUSSION

The group I (control) and group II (treated with CdCl₂) resealed embryonated chicken eggs were unfasten the cellophane tape and examined following 72 h of incubation after treatment. Modulatory effects of CdCl₂ salt on the process of angiogenesis were observed by visualizing the gross and histopathological alterations in chorioallantoic membranes (CAM).

Gross examination of control group I revealed well arbourized vasculature with major and minor blood vessels (Fig. 1a) where the treated group II eggs showed reduction in number of secondary and tertiary blood vessel (Fig. 1b).

Histopathological analysis also corroborated the gross examinations of the CAM, which revealed normal thickness of CAM among controls (Group I) with concordant dispersed Mesodermal Blood Vessels (MBV) with loose connective tissue (Fig. 1c). However, in CdCl₂ salt treated CAM, mesodermal blood vessels were found to be breached out of which RBCs were oozing out. Also occasional abnormal thickening and accumulation of densely arranged cells at chorionic and allantoic sides was observed (Fig. 1d).

At present, chemotherapeutic drugs are being mostly used to treat cancer as well as other diseases. Unfortunately, many compounds showed limited efficacy with impaired delivery, penetration and specificity for the cancerous cells, hence causing deleterious side effects¹⁶. The activity of these compounds is mainly restricted by the drug resistance towards tumor cells.

Fig. 1(a-d):

(a) Gross examination of CAM of control eggs (Group I) with well arborized vascular system, (b) CAM of CdCl2 treated eggs (Group II) showing primary blood vessel with less visible secondary and tertiary blood vessels, (c) Histopathology of CAM of controls (Group I, 100X) showing AE and CE of uniform thickness and (d) CdCl2 treated (Group II, 400X) CAM showing abnormal thickening and accumulation of densely arranged cells at chorionic and allantoic sides with and occasional bleeding; AE: Allantoic epithelium, CE: Chorionic epithelium, MBV: Mesodermal blood vessels

Pre-clinical studies and clinical trials suggest that angiogenesis-based therapy may be useful in the future care of patients¹⁷. In particular, anti-angiogenic therapy is a unique approach to destroy tumor cells, since it inhibits the growth of blood vessels¹⁸, it does not target cancer cells directly. So far, anti-angiogenic agents are not likely to result in bone marrow suppression, gastrointestinal adverse symptoms or hair loss like other anti cancer therapies. This approach to slow the growth of blood vessels may require several months to a year, thus, the administration of the agents at lower doses and longer uninterrupted periods than the usual doses and periods of conventional cytotoxic agents should be considered in the design, several of these are under clinical trials^19,20. The development of resistance to angiogenic inhibitors has not been a big problem so far, furthermore, a combination of anti-angiogenic therapy and conventional therapy may be more effective than either therapy alone^21,22, which may provide a novel, selective, safe and reasonable treatment in future medicine.

Cadmium (Cd) exposure brings changes in the blood vessel architecture and can cause pathologiocal conditions. It induces changes in morphological features and is through altering the mRNA and protein level expression. The results of present observations are in concordant with the findings of the Woods et al.²³ and Ma and Waxman²², that it inhibits the tube formation. In this results too, it is demonstrated that number of secondary and tertiary blood vessels are reduced in number. With Cd, c-Jun N-terminal kinases (JNKs)²⁴ and VEGF²⁵ are activated, which can be related to the increased angiogenesis, actually it reduce angiogenesis by virtue of inhibition in tube formation, which indicate that Cd is involved in anti-angiogenesis using diverse molecular pathways. Also cadmium (II) complex with selenosemicarbazone potentially inhibit NO production in human endothelial cell leading to anti-angiogenesis²⁵.

Hence, due to the great anti-angiogenic effect all are reduces the number of blood vessels also affected the endothelial layer, therefore it has great role in therapeutics of cancer and tumor related disease.

CONCLUSION

The CdCl₂ activate extracellular signal-regulated kinases (ERK) and AKT signaling with induction of hypoxia-inducible factor-1 (HIF-1), a key pro-angiogenic molecule. Also it induce vascular endothelial growth factor expression, however despite inducing angiogenic factors, reduction in endothelial tube formation and NO production, exhibits its complex impact on process of angiogenesis. However, its anti-angiogenic property shown in this study, as exhibited by reduced number of secondary and tertiary blood vessels with occasional breaching of blood vessels indicated its therapeutic value in cancer therapy but it require further dose optimization and safety testing. Present study may pave road for further study in direction of exploitation of antiangiogenic property of Cd.

SIGNIFICANCE STATEMENT

The effects of CdCl₂ were found to be anti-angiogenic on CAM evident by reduction in the number of secondary and tertiary blood vessels and revealed detrimental effects by inducing inflammation. Therefore, it may have potential to be explored as a therapeutic material in anti-angiogenic and wound repair medicines.

ACKNOWLEDGMENTS

Assistance from DBT builder Programme is gratefully acknowledged. Authors of the manuscript thank and acknowledge their respective Universities/ Institutes.