**3. Results**

## **3.1 A1 (reactive) and A2 (normal) HFAs in culture**

The results show that the HFAs increase in size and number with increasing time in tissue culture. Distinct differences were observed in the morphology of the cells present in A1 and A2 flasks, for example A1 astrocytes have more fibroblast-like structures as compared to A2 astrocytes, which look more like neurons (**Figure 7**). A noticeable difference was observed in the thickness of the processes between the two types of the HFAs. This thickening was due to the accumulation of filament proteins such as glial fibrillary acidic proteins (GFAP) as shown in **Figure 8**. Whereas **Figure 9** shows the astrocytes present in the spontaneously hypertensive rat (SHR) and normal Wistar Kyoto (WKY) rat brain slices. **Figure 9**, shows the similarities between A1 HFAs and the SHR astrocytes, implying that the A1 HFAs mimic the hypertensive condition.

### **3.2 Identification of proteins in A1 and A2 HFAs, using LC/MS/MS technique**

The present study was focused on specific proteins, such as glial fibrillary acidic protein (GFAP), calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK).

## *3.2.1 GFAP*

The GFAP protein is an indicator of HFAs reactivity, therefore, it was important to carefully measure the area under the curve to determine its degree of intensity. For comparative analysis, the peaks closest to the center of the peptides-spectrum matches (PSMS) were selected, against part per million (ppm) as shown above in **Figure 6**.

The elevated level of GFAP in A1 cells (4.39 ± 0.4) was observed, compared to A2 cells (3.02 ± 0.3), as shown in **Figure 9**. A significantly higher level of GFAP (P < 0.05) in A1 compared to A2, resulted in the thickening of processes (indicated by arrows in **Figures 7, 9** and **10** above).

Protein molecules such as calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK) are known to be activated by intracellular calcium, leading to their relevant cell signalling cycles and in turn gene [24] modifications.

**Figure 7.**

*Shows normal HFAs in two flasks, labelled A1 and A2. Panel A and B display 2 and 10 days old normal HFAs, respectively. Panel C, represents 18 days old normal (A2) and ATP-treated reactive (A1) astrocytes, arrows indicating the thickening of the internal filaments. Scale bar, 100 μm.*

#### **Figure 8.**

*Panel A, shows the normal astrocytes in WKY rat brain slices. Panel B, displays the reactive astrocytes in SHR brain slices. Arrows indicate the upregulation of intermediate filament proteins in SHR astrocytes. Panel C, shows the difference between the intensity of GFAP in the astrocytes of normal and hypertensive rats. \*\*\*P < 0.001, scale bar, 20 μm.*

**67**

*3.2.2 Calpain*

**Figure 10.**

**Figure 9.**

*3.2.3 Calpastatin*

ing to cytotoxicity in reactive HFAs.

*on day 10 it was 2.5* μ*m as compared to 4.5 μm on day 18. Scale bar, 10 μm.*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

The results show a significantly higher level (**Figure 11**) of Calpain in A1 (6.37 ± 0.6) compared to A2 (3.40 ± 0.2), indicating a greater level of calpain lead-

*Panel A (reactive) and panel B (normal) show HFAs after 10 days in culture. Panel C (reactive) and panel D (normal) is after 18 days in culture. Arrows indicate the difference in the thickening of internal filaments, i.e.,* 

*Level of glial fibrillary acidic protein (GFAP) in normal and reactive astrocytes. Area under the curve (AUC)* 

The results indicate that reactive astrocytes A1 (5.52 ± 0.5) have significantly higher levels of calpastatin as compared to the normal A2 (4.09 ± 0.6) astrocytes

*DOI: http://dx.doi.org/10.5772/intechopen.98561*

*values are shown as means ± SEM, \*P < 0.05, (n = 9).*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes DOI: http://dx.doi.org/10.5772/intechopen.98561*

#### **Figure 9.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

by arrows in **Figures 7, 9** and **10** above).

The GFAP protein is an indicator of HFAs reactivity, therefore, it was important to carefully measure the area under the curve to determine its degree of intensity. For comparative analysis, the peaks closest to the center of the peptides-spectrum matches (PSMS) were selected, against part per million (ppm) as shown above in **Figure 6**. The elevated level of GFAP in A1 cells (4.39 ± 0.4) was observed, compared to A2 cells (3.02 ± 0.3), as shown in **Figure 9**. A significantly higher level of GFAP (P < 0.05) in A1 compared to A2, resulted in the thickening of processes (indicated

Protein molecules such as calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK) are known to be activated by intracellular calcium, leading to their relevant cell signalling cycles and in turn gene [24]

*Panel A, shows the normal astrocytes in WKY rat brain slices. Panel B, displays the reactive astrocytes in SHR brain slices. Arrows indicate the upregulation of intermediate filament proteins in SHR astrocytes. Panel C, shows the difference between the intensity of GFAP in the astrocytes of normal and hypertensive rats.* 

*Shows normal HFAs in two flasks, labelled A1 and A2. Panel A and B display 2 and 10 days old normal HFAs, respectively. Panel C, represents 18 days old normal (A2) and ATP-treated reactive (A1) astrocytes, arrows* 

*indicating the thickening of the internal filaments. Scale bar, 100 μm.*

*3.2.1 GFAP*

modifications.

**66**

**Figure 8.**

**Figure 7.**

*\*\*\*P < 0.001, scale bar, 20 μm.*

*Level of glial fibrillary acidic protein (GFAP) in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ± SEM, \*P < 0.05, (n = 9).*

#### **Figure 10.**

*Panel A (reactive) and panel B (normal) show HFAs after 10 days in culture. Panel C (reactive) and panel D (normal) is after 18 days in culture. Arrows indicate the difference in the thickening of internal filaments, i.e., on day 10 it was 2.5* μ*m as compared to 4.5 μm on day 18. Scale bar, 10 μm.*

#### *3.2.2 Calpain*

The results show a significantly higher level (**Figure 11**) of Calpain in A1 (6.37 ± 0.6) compared to A2 (3.40 ± 0.2), indicating a greater level of calpain leading to cytotoxicity in reactive HFAs.

#### *3.2.3 Calpastatin*

The results indicate that reactive astrocytes A1 (5.52 ± 0.5) have significantly higher levels of calpastatin as compared to the normal A2 (4.09 ± 0.6) astrocytes

#### **Figure 11.**

*Level of calpain in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ± SEM, \*\*\*P < 0.001, (n = 9).*

#### **Figure 12.**

*Level of calpastatin in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ± SEM, \*\*P < 0.01, (n = 9).*

(**Figure 12**). Calpastatin is the most specific endogenous calpain-inhibitor, which binds to the active sites of calpain to prevent its activation [25].

#### *3.2.4 Cathepsin*

A highly significant increase was observed in the levels of cathepsin in A1 (6.31 ± 0.7) compared to A2 (3.86 ± 0.4) cells (**Figure 13**). Both types of HFAs display human specific cathepsin.

#### *3.2.5 Mitogen activated protein kinase (MAPK)*

SP3 Proteomic results show a significantly higher level of MAPK enzyme in A1 (6.31 ± 0.7) compared to A2 (3.72 ± 0.4) HFAs (**Figure 14**). This kinase is an essential component of the cell signalling pathway, responsible for the communication between a receptor on the cell-surface to the DNA inside the nucleus [6, 26].

**69**

**4. Discussion**

*means ± SEM, \*\*\*P < 0.001, (n = 9).*

**Figure 14.**

**Figure 13.**

*means ± SEM, \*\*\*P < 0.001, (n = 9).*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

*Level of cathepsin in normal and reactive astrocytes. Area under the curve (AUC) values are shown as* 

All the above-mentioned proteins were identified in both A1 and A2 HFAs, using immunocytochemistry and proteomics (SP3) protocols. Higher levels of calcium-

*Level of MAPK in normal and reactive astrocytes. Area under the curve (AUC) values are shown as* 

This study demonstrated for the first time that reactive astrocytes in HFAs mimic hypertensive conditions. The present research indicates that the reactive (A1) HFAs have a similar protein profile to astrocytes of hypertensive rats. ATP was used in these experiments to mimic endogenous conditions, as it is released from

The proteins selected as predictive biomarkers for HTN, are either responsible for the reactivity of the astrocytes, or produced because of the interaction between

gated proteins in A1 are potential biomarkers of HTN.

the damaged cells following tissue injury [27, 28].

*DOI: http://dx.doi.org/10.5772/intechopen.98561*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes DOI: http://dx.doi.org/10.5772/intechopen.98561*

#### **Figure 13.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

(**Figure 12**). Calpastatin is the most specific endogenous calpain-inhibitor, which

*Level of calpastatin in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means* 

*Level of calpain in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ±* 

A highly significant increase was observed in the levels of cathepsin in A1 (6.31 ± 0.7) compared to A2 (3.86 ± 0.4) cells (**Figure 13**). Both types of HFAs

SP3 Proteomic results show a significantly higher level of MAPK enzyme in A1 (6.31 ± 0.7) compared to A2 (3.72 ± 0.4) HFAs (**Figure 14**). This kinase is an essential component of the cell signalling pathway, responsible for the communication between a receptor on the cell-surface to the DNA inside the nucleus [6, 26].

binds to the active sites of calpain to prevent its activation [25].

**68**

*3.2.4 Cathepsin*

*± SEM, \*\*P < 0.01, (n = 9).*

**Figure 12.**

**Figure 11.**

*SEM, \*\*\*P < 0.001, (n = 9).*

display human specific cathepsin.

*3.2.5 Mitogen activated protein kinase (MAPK)*

*Level of cathepsin in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ± SEM, \*\*\*P < 0.001, (n = 9).*

#### **Figure 14.**

*Level of MAPK in normal and reactive astrocytes. Area under the curve (AUC) values are shown as means ± SEM, \*\*\*P < 0.001, (n = 9).*

All the above-mentioned proteins were identified in both A1 and A2 HFAs, using immunocytochemistry and proteomics (SP3) protocols. Higher levels of calciumgated proteins in A1 are potential biomarkers of HTN.

### **4. Discussion**

This study demonstrated for the first time that reactive astrocytes in HFAs mimic hypertensive conditions. The present research indicates that the reactive (A1) HFAs have a similar protein profile to astrocytes of hypertensive rats. ATP was used in these experiments to mimic endogenous conditions, as it is released from the damaged cells following tissue injury [27, 28].

The proteins selected as predictive biomarkers for HTN, are either responsible for the reactivity of the astrocytes, or produced because of the interaction between orexin and NMDA receptors. Most of the proteins selected as predictive biomarkers were calcium binding proteases. The results from A1 and A2 HFAs show that calcium dependent proteins, such as GFAP, calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK) were present at higher concentrations in reactive (A1) than in non-reactive (A2) astrocytes. These results suggest that the above-mentioned proteins may be targeted as therapeutic agents for the prevention of HTN.

Immunocytochemistry, in conjunction with the confocal microscopy were used to assess and visualise the proteins, such as internal filament proteins in HFAs. Mass spectrometry (LC/MS/MS) was used to validate the results, obtained by using immunocytochemistry. The SP3 proteomic protocol was employed to identify and estimate the protein-peptide molecules.

Reactive astrocytes (A1) were prepared from the normal (A2) HFAs, by using ATP (**Figure 7**), as described earlier in primary cell lines of neonatal rats [29]. In contrast, a recent research [23] reported that ATP was not sufficient to induce a complete reactive phenotype of astrocytes, in vitro. This discrepancy between the two results may be because HFAs were used in the current study, whereas Adzic et al. [23] used mature astrocytes obtained from the rats. This concept was further verified by the micrographs from light microscopy (**Figure 7**) and confocal microscopy (**Figures 8** and **10**) which showed a gradual increase in the confluency as well as in the thickness of the processes of the HFAs with increasing time in culture. Hence, our experiments confirmed that the reactivity profile of the astrocytes changed as they developed. This proliferation of the filaments is due to an increase in the accumulation of GFAP with time [30]. In A1 astrocytes, the thickness of the processes on day 10 was 2.5 μm as compared to 4.5 μm on day 18 (**Figures 7** and **10**), indicating that there was a periodic increase in the concentration of GFAP in the reactive astrocytes. This significant thickening of astrocytic processes in A1 HFAs (**Figure 8**), was like that observed in the astrocytes of hypertensive rats (**Figure 9**). SP3 proteomic result for GFAP (**Figure 9**) was similar to the microscopic result, representing a significant increase of GFAP in reactive as compared to normal astrocytes. These results of the protein analysis were similar to the study by Hol et al. [31], indicating that an upregulation of GFAP in reactive astrocytes, coincides with neurodegenerative diseases such as HTN. Similarly, a previous study [32] has established that increased levels of GFAP was directly related to hypertension, in SHRs. Hence, based on the literature and our study results, it can be concluded that the reactivity in astrocytes could be a direct indicator of hypertension.

The results of our study showed that A1 has both increased levels of GFAP (**Figure 9**) and calpain (**Figure 11**), indicating the increased reactivity in A1 astrocytes may be due to the higher level of calpain, as shown in the previous studies [33, 34], resulting in atherosclerosis and hence HTN [35]. Other studies [36] indicate that calpain can also act on the extracellular substrates, such as collagen-fibronectin, to modulate the cell activity. As calpain can act both intra and extracellularly, it could be a therapeutic target for the prevention of cardiovascular diseases, including hypertension.

In contrast, calpastatin, an endogenous inhibitor of calpain, attenuates its cytotoxicity, thus increasing the levels of calpastatin may be beneficial in the regulation of HTN [25]. The current results show lower expression of calpastatin (**Figure 12**) as compared to calpain (**Figure 11**) in A1 astrocytes, indicating a decreased level of calpastatin in reactive HFAs. These results also suggest that an additional amount of exogenous calpastatin may decrease the toxic activity of calpain, by blocking the active sites of calpain [37]. This remarkable role of calpastatin makes it a promising therapeutic agent for managing blood pressure.

Likewise, another endogenous protease cathepsin, which is involved in inflammatory disorders, was found to be significantly higher (**Figure 13**) in reactive

**71**

**5. Conclusion**

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

(A1) astrocytes. Evidence suggests that cathepsin plays an important role in HTN through vascular modulation [38], leading to atherosclerosis. It has also been reported that cathepsin regulates the phosphorylation of mitogen activated protein kinase- kinase (MEK) in Angiotensin II-dependent hypertension [39]. Angiotensin II induces g-protein regulated MAPK cell signalling cycle [40], therefore MAPK was measured in both A1 and A2 astrocytes (**Figure 14**), and it was found to be significantly higher in A1 as compared to A2. This augmented level of MAPK in A1 HFAs could be responsible for the thickening of their intermediate filaments (**Figure 10**), as indicated previously in another study [41] that MAPK upregulates

In a recent study, the phosphorylation of MAPK in the arteries of hypertensive patients as well as in a mice model [39] was observed. Moreover, the proliferation of arterial smooth muscle cells in both, human and mice models were detected. Thus, a

Our initial findings based on immunocytochemistry and SP3 proteomic results

Protein-based therapeutic agents have been highly successful in clinics [44]. More than hundred original and modified therapeutic proteins are used up till now.

Further experiments are needed to test these enzymes, using ELISA kits for the specific regulatory proteins, such as S100B, soluble receptor for advanced glycation end product (sRAGE) and GFAP, to identify the most promising predictive biomarker for HTN. The highly specific indicator of astrocytic reactivity, GFAP [50], was measured earlier by immunocytochemistry, proteomics, and now ELISA protocol will be used for further quantification of cytotoxicity due to the higher

Similarly, the Ca2+-binding protein S100B, acts both intra and extracellularly in HFAs. Inside the cell, S100B acts as a stimulator of proliferation and activation of astrocytes. Whereas, extracellularly, S100B engages sRAGE in pro-proliferative activities [51]. Though, sRAGE might not be the only S100B receptor, and S100B's ability to engage sRAGE might be regulated by its interaction with other calcium-

This study shows for the first time that reactive astrocytes in HFAs mimic hypertensive conditions, and calcium-dependent proteins such as GFAP, calpain,

binding proteases, such as calpain, calpastatin, cathepsin and MAPK.

There are at least five ways of utilising proteins as therapeutic agents:

c.interfering with an intermediate molecule or its function [47]

d.providing synthetic molecules for normal function [48]

vascular modulatory role of MAPK, which is related to HTN, was confirmed.

indicate that identifying molecules such as calpain, calpastatin, cathepsin and MAPK may be useful in reducing the reactivity in the astrocytes, which is an indicator of HTN. The present study shows that in A1, there is an increased levels of calcium gated proteins, possibly due to higher concentration of the intracellular

*DOI: http://dx.doi.org/10.5772/intechopen.98561*

calcium [42, 43], leading to hypertension.

a. replacing damaged proteins [45]

b.augmenting the effects of weak proteins [46]

e.delivering other compounds or proteins [49]

concentration of GFAP in the reactive astrocytes.

cell proliferation.

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes DOI: http://dx.doi.org/10.5772/intechopen.98561*

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

estimate the protein-peptide molecules.

indicator of hypertension.

orexin and NMDA receptors. Most of the proteins selected as predictive biomarkers were calcium binding proteases. The results from A1 and A2 HFAs show that calcium dependent proteins, such as GFAP, calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK) were present at higher concentrations in reactive (A1) than in non-reactive (A2) astrocytes. These results suggest that the above-mentioned

Immunocytochemistry, in conjunction with the confocal microscopy were used

Reactive astrocytes (A1) were prepared from the normal (A2) HFAs, by using ATP (**Figure 7**), as described earlier in primary cell lines of neonatal rats [29]. In contrast, a recent research [23] reported that ATP was not sufficient to induce a complete reactive phenotype of astrocytes, in vitro. This discrepancy between the two results may be because HFAs were used in the current study, whereas Adzic et al. [23] used mature astrocytes obtained from the rats. This concept was further verified by the micrographs from light microscopy (**Figure 7**) and confocal microscopy (**Figures 8** and **10**) which showed a gradual increase in the confluency as well as in the thickness of the processes of the HFAs with increasing time in culture. Hence, our experiments confirmed that the reactivity profile of the astrocytes changed as they developed. This proliferation of the filaments is due to an increase in the accumulation of GFAP with time [30]. In A1 astrocytes, the thickness of the processes on day 10 was 2.5 μm as compared to 4.5 μm on day 18 (**Figures 7** and **10**), indicating that there was a periodic increase in the concentration of GFAP in the reactive astrocytes. This significant thickening of astrocytic processes in A1 HFAs (**Figure 8**), was like that observed in the astrocytes of hypertensive rats (**Figure 9**). SP3 proteomic result for GFAP (**Figure 9**) was similar to the microscopic result, representing a significant increase of GFAP in reactive as compared to normal astrocytes. These results of the protein analysis were similar to the study by Hol et al. [31], indicating that an upregulation of GFAP in reactive astrocytes, coincides with neurodegenerative diseases such as HTN. Similarly, a previous study [32] has established that increased levels of GFAP was directly related to hypertension, in SHRs. Hence, based on the literature and our study results, it can be concluded that the reactivity in astrocytes could be a direct

The results of our study showed that A1 has both increased levels of GFAP (**Figure 9**) and calpain (**Figure 11**), indicating the increased reactivity in A1 astrocytes may be due to the higher level of calpain, as shown in the previous studies [33, 34], resulting in atherosclerosis and hence HTN [35]. Other studies [36] indicate that calpain can also act on the extracellular substrates, such as collagen-fibronectin, to modulate the cell activity. As calpain can act both intra and extracellularly, it could be a therapeutic

In contrast, calpastatin, an endogenous inhibitor of calpain, attenuates its cytotoxicity, thus increasing the levels of calpastatin may be beneficial in the regulation of HTN [25]. The current results show lower expression of calpastatin (**Figure 12**) as compared to calpain (**Figure 11**) in A1 astrocytes, indicating a decreased level of calpastatin in reactive HFAs. These results also suggest that an additional amount of exogenous calpastatin may decrease the toxic activity of calpain, by blocking the active sites of calpain [37]. This remarkable role of calpastatin makes it a promising

Likewise, another endogenous protease cathepsin, which is involved in inflam-

matory disorders, was found to be significantly higher (**Figure 13**) in reactive

target for the prevention of cardiovascular diseases, including hypertension.

therapeutic agent for managing blood pressure.

proteins may be targeted as therapeutic agents for the prevention of HTN.

to assess and visualise the proteins, such as internal filament proteins in HFAs. Mass spectrometry (LC/MS/MS) was used to validate the results, obtained by using immunocytochemistry. The SP3 proteomic protocol was employed to identify and

**70**

(A1) astrocytes. Evidence suggests that cathepsin plays an important role in HTN through vascular modulation [38], leading to atherosclerosis. It has also been reported that cathepsin regulates the phosphorylation of mitogen activated protein kinase- kinase (MEK) in Angiotensin II-dependent hypertension [39]. Angiotensin II induces g-protein regulated MAPK cell signalling cycle [40], therefore MAPK was measured in both A1 and A2 astrocytes (**Figure 14**), and it was found to be significantly higher in A1 as compared to A2. This augmented level of MAPK in A1 HFAs could be responsible for the thickening of their intermediate filaments (**Figure 10**), as indicated previously in another study [41] that MAPK upregulates cell proliferation.

In a recent study, the phosphorylation of MAPK in the arteries of hypertensive patients as well as in a mice model [39] was observed. Moreover, the proliferation of arterial smooth muscle cells in both, human and mice models were detected. Thus, a vascular modulatory role of MAPK, which is related to HTN, was confirmed.

Our initial findings based on immunocytochemistry and SP3 proteomic results indicate that identifying molecules such as calpain, calpastatin, cathepsin and MAPK may be useful in reducing the reactivity in the astrocytes, which is an indicator of HTN. The present study shows that in A1, there is an increased levels of calcium gated proteins, possibly due to higher concentration of the intracellular calcium [42, 43], leading to hypertension.

Protein-based therapeutic agents have been highly successful in clinics [44]. More than hundred original and modified therapeutic proteins are used up till now. There are at least five ways of utilising proteins as therapeutic agents:

a. replacing damaged proteins [45]

b.augmenting the effects of weak proteins [46]

c.interfering with an intermediate molecule or its function [47]

d.providing synthetic molecules for normal function [48]

e.delivering other compounds or proteins [49]

Further experiments are needed to test these enzymes, using ELISA kits for the specific regulatory proteins, such as S100B, soluble receptor for advanced glycation end product (sRAGE) and GFAP, to identify the most promising predictive biomarker for HTN. The highly specific indicator of astrocytic reactivity, GFAP [50], was measured earlier by immunocytochemistry, proteomics, and now ELISA protocol will be used for further quantification of cytotoxicity due to the higher concentration of GFAP in the reactive astrocytes.

Similarly, the Ca2+-binding protein S100B, acts both intra and extracellularly in HFAs. Inside the cell, S100B acts as a stimulator of proliferation and activation of astrocytes. Whereas, extracellularly, S100B engages sRAGE in pro-proliferative activities [51]. Though, sRAGE might not be the only S100B receptor, and S100B's ability to engage sRAGE might be regulated by its interaction with other calciumbinding proteases, such as calpain, calpastatin, cathepsin and MAPK.

### **5. Conclusion**

This study shows for the first time that reactive astrocytes in HFAs mimic hypertensive conditions, and calcium-dependent proteins such as GFAP, calpain, calpastatin, cathepsin and MAPK could be considered as potential predictive biomarkers for HTN.
