**1. Introduction**

Scientific studies have established that prevalence of hypertension (HTN) needs to be reduced in order to control cardiovascular and cerebrovascular diseases [1]. At present, many laboratories around the world are exploring a plethora of biomarkers, as early indicators of HTN [2, 3], i.e., triglycerides, C-reactive protein, fibrinogen, serum albumin, uric acid, homocysteine and intracellular adhesion molecule-1(ICAM-1) [4]. All these biomarkers for hypertension are produced as a result of chronic disease-related comorbidities such as atherosclerosis, Type 2 diabetes and renal failure, therefore, they cannot be considered as 'true' predictive biomarkers for hypertension. Hence, this study is focusing on glial fibrillary acidic protein (GFAP), and a group of calcium-dependent proteases, such as calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK), known to be strongly associated with HTN; hence could be potential early biomarkers for HTN.

Similarly, an endogenous chemical, N-Methyl-D-Aspartic acid (NMDA), a type of glutamate is also documented as linked with HTN [5]. NMDA is a major excitatory neurotransmitter in the central nervous system, consisting of two types of receptors namely synaptic and extrasynaptic. Research indicates that the activation of the synaptic NMDA receptors is neuroprotective; whereas, the stimulation of extrasynaptic NMDA receptors (NMDARs) promotes cell death [6] and resultant HTN, which can be regulated by NMDA antagonists, such as 1-amino cyclo propane carboxylic acid (ACPC) [7]. NMDA type glutamate receptors are not only present in the neuronal cells but also in the non-neuronal cells such as astrocytes [8]. Recent studies have demonstrated a significant role of astrocytes in regulating blood flow due to the elevation in intracellular calcium (Ca2+) [9], which plays a significant role in the regulation of blood pressure.

Astrocytes are present between blood vessels and neurons and are responsible for changes in the arterial blood pressure [10]. A decline in the cerebral blood supply activates the astrocytes to release a chemical signal to the nearby neurons that raises blood pressure, restoring blood flow and oxygen supply to the brain [10]. Thus, astrocytes perform a balancing role between brain perfusion and neuronal activities by mobilising their internal calcium [11], which in turn triggers the release of chemical transmitters such as glutamate [12]. Consequently, there is a calciumdependent bidirectional signalling pathway between astrocytes and neurons [13], which opens up the possibility of astrocytic involvement in the modulation of calcium-dependent molecules such as calpain, calpastatin, cathepsin and MAPK, which can potentially be considered as direct predictive biomarkers of HTN.

Certain behavioural conditions, such as, stress also elevates blood pressure. Under stressful conditions, excessive release of corticotropin-releasing hormone (CRH) activates NMDA receptors, resulting in an influx of Ca2+ molecules, which enhance the activity of m-calpain [14]. Calpain is one of the major calcium-dependent proteolytic enzymes with various isoforms such as μ-calpain and m-calpain, which are activated by the synaptic and extra-synaptic NMDA receptors respectively [15, 16]. Studies have indicated that the activation of μ-calpain is important for cell-survival, whereas the stimulation of m-calpain initiates toxic effects and cell death [6], due to its interaction with NMDAR [17]. Activation of the type of NMDA receptor defines the communication from the synapse to the nucleus [18].

Stimulation of synaptic NMDAR phosphorylates the intermediate filament proteins by extracellular signal regulated kinases 1 and 2 (ERK1/2) [19]. However, the activation of extrasynaptic NMDAR fails to phosphorylate and translocate intermediate filament proteins into the nucleus. The phosphorylated or non-phosphorylated state of intermediate filament proteins determine whether it promotes cell survival or induces cell death. These diverse functions that require the regulation

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*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

of gene and synapse-to-nucleus communications are controlled by nuclear calcium signalling [20]. Overexpression of these proteins induces neurodegeneration, whereas, its suppression has the opposite effect, therefore, the molecules that keep

To date, most proteomic research, targeted at discovering biomarkers, has failed to incorporate adequate biomarker validation studies in independent sample sets. These are necessary steps in the translation of potential biomarkers into clinical practice. Recent development of sophisticated mass spectrometry-based quantitation of multiple proteins has enabled the validation of candidate biomarkers in

The literature indicates that reactive astrocytes (A1) and their intermediate filaments contain greater concentrations of glial fibrillary acidic protein (GFAP) [21], compared to normal astrocytes (A2). The purpose of this study was, to identify and quantify the differences between the proteins of interest, present in the reactive and the normal human foetal astrocytes (HFAs), using immunocytochemistry and single-pot, solid-phase-enhanced sample-preparation (SP3) [22] proteomic techniques. We aim to detect the immunocytochemical differences between the A1 and A2 HFAs using GFAP antibodies and to identify a panel of protein biomarkers in A1

HFAs were obtained from the Biobank of Macquarie University, after approval

After getting a material transfer agreement (MTA # 17/979) between the two universities, the HFAs were tested for mycoplasma contamination and were found to be negative. The HFAs were then cultured in T75 flasks using Roswell Park Memorial Institute (RPMI 1640) media containing 10% heat-inactivated foetal calf serum (FCS) (**Table 1**), at 37°C, in a 5% CO2 humidified incubator. Freshly

cells/T75-cm2

RPMI (4.5 g/L glucose, L-glutamine, and 25 mM HEPES buffer), plus 10% FCS at

For re-seeding, HFAs were detached from the flask by using 4 ml of trypsin, incubated for 3 minutes at 37°C, in a 5% CO2 humidified incubator. After incubation with trypsin, the cells were transferred to a 15 ml falcon tube and were centrifuged at 1600 g rpm, for 3 minutes. Trypsin was removed by washing three times with phosphate buffered saline (PBS). The cells were then resuspended in 2 ml of fresh media by gentle mixing and 1 ml of this cell solution was added to each T75 flask with 14 ml

RPMI, labelled as A1 and A2 for reactive and normal astrocytes, respectively.

After achieving 95 to 100% confluency and three days before the proteomics experiment, the HFAs were incubated in the RPMI media with 1% FCS instead of 10% FCS, to avoid false positive proteomic results due to the proteins present in the 10% FCS. Twenty-four hours before the pellet formation for SP3 [22] protocol,

**2.3 Conversion of normal HFAs (A2) into reactive HFAs (A1)**

in tissue culture flasks containing

from the UTS Human Research Ethics Committee (ETH17–1883).

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

the balance may act as therapeutic targets for HTN.

different biological materials, such as astrocytes.

and A2 HFAs for the prediction of hypertension.

**2.2 Protocol for the cell culture of primary HFAs**

prepared media was used to feed the cells every five days.

37°C, and incubated in a 5% CO2 humidified incubator.

The HFAs were seeded at 4x107

**2. Materials and methods**

**2.1 Ethical approval**

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

of gene and synapse-to-nucleus communications are controlled by nuclear calcium signalling [20]. Overexpression of these proteins induces neurodegeneration, whereas, its suppression has the opposite effect, therefore, the molecules that keep the balance may act as therapeutic targets for HTN.

To date, most proteomic research, targeted at discovering biomarkers, has failed to incorporate adequate biomarker validation studies in independent sample sets. These are necessary steps in the translation of potential biomarkers into clinical practice. Recent development of sophisticated mass spectrometry-based quantitation of multiple proteins has enabled the validation of candidate biomarkers in different biological materials, such as astrocytes.

The literature indicates that reactive astrocytes (A1) and their intermediate filaments contain greater concentrations of glial fibrillary acidic protein (GFAP) [21], compared to normal astrocytes (A2). The purpose of this study was, to identify and quantify the differences between the proteins of interest, present in the reactive and the normal human foetal astrocytes (HFAs), using immunocytochemistry and single-pot, solid-phase-enhanced sample-preparation (SP3) [22] proteomic techniques. We aim to detect the immunocytochemical differences between the A1 and A2 HFAs using GFAP antibodies and to identify a panel of protein biomarkers in A1 and A2 HFAs for the prediction of hypertension.
