Expression of mRNA vascular endothelial growth factor in hypospadias patients

Background Hypospadias is a relatively common genital anomaly in humans, usually followed by inelastic dartos that causes penile chordee. Vascular endothelial growth factor (VEGF) is strongly linked to the viscoelasticity of tissues and their elastic phase. This study aimed to evaluate VEGF expressions in (1) fascia dartos between hypospadias and controls and (2) chordee severity. Methods This prospective cohort study involved 65 specimens from patients with hypospadias and ten specimens from controls. The samples were analyzed by quantitative real-time polymerase chain reaction (qPCR) for VEGF expression. Results The expressions of VEGF were not different between proximal and distal hypospadias patients and controls (fold change: distal − 0.25; fold change: proximal − 0.2; p = 0.664). The scaled expressions related to chordee severity were mild − 0.1; moderate 0.1; severe − 0.25 (p = 0.660). Conclusions VEGF expressions might not affect the severity of hypospadias and chordee, implying the pathogenesis is complex involving many growth factors. Further study with a larger sample size is necessary to clarify and confirm our findings.


Introduction
Hypospadias, a relatively common human genital abnormality, is a urethral opening disorder that is not positioned correctly at the tip of the penis. Hypospadias is the second most common congenital anomaly in boys and the most common type of penis deformity [1,2]. Molecular processes are needed in the occurrence of hypospadias, but the relationships and functions of these two elements are still unknown [3]. The exact cause of hypospadias has been identified in about 20% of cases, especially in the most challenging conditions [1].
Hypospadias may be related to an abnormality of the dartos fascia. However, this hypothesis is not supported by solid evidence from histopathology. There are contrasting studies of histopathological aspects of connective tissue in patients with hypospadias. The dartos fascia of hypospadias had abnormal tissue that gave it the thick and inelastic character [4,5]. Inelastic dartos fascia tissue in patients diagnosed with hypospadias is a pathological tissue [4,6,7].
VEGF regulates elastic modulus and increases matrix stiffness in cardiomyocytes [8]. It is strongly associated with tissue viscoelasticity and the elastic cervix phase shortening [9]. In addition, VEGF plays a pivotal part in endothelial cells' proliferation, migration, and differentiation [10]. VEGF induces α1β1 and α2β1 integrins expression in microvascular endothelial cells [11], endothelial cell migration, and proliferation [12,13]. VEGF is not retained intra-cellularly but attaches to the cell surface or Open Access  [14]. However, the role of VEGF on dartos tissue remains unknown. Hence, this study aimed to investigate the impact of VEGF modulated dartos on tissue elasticity.

Patients
A prospective cohort study was performed in 65 patients who underwent repair of hypospadias with chordee without DSD, and ten patients underwent circumcision as controls between 2018 and 2020. The samples were then divided into three groups: distal hypospadias, proximal hypospadias, and controls. We used periurethral dartos tissues collected during excision of chordee. The Ethical Committee of our university approved this study(KE/0287/03/2020), and written informed consent to participate in this study was obtained from all the parents.

qPCR analysis
All dartos tissue of each patient was placed in a single tube. All samples were homogenized. Total RNA was extracted using Hybrid-R ™ Isolation Kit, and cDNA was extracted from 200 ng of total RNA using NEXpro ™ qRT-PCR Kit. Gene expression analysis by Quantitative PCR has been used for VEGF (F: 5'-CCC ACT GAG GAG TCC AAC AT-3′ and R: 5′-AAA TGC TTT CTC CGC TCT GA-3′), and GAPDH (F: 5′-GCA TCC TGG GCT ACA CTG AG-3′ and R: 5′-TCC ACC ACC CTG TTG CTG TA-3′) used as internal control. Conditions for amplification consisted of an initial denaturing phase at 95 °C for 10 min, proceeded by 40 cycles at 95 °C for 20 secs, at 56 °C for 40 secs, and 72 °C for 60 secs. Extension done at 72 °C for 5 min. The qPCR amplified samples were analyzed by the BiONEER Exicycle ™ 96 (BioNEER, Daejeon, South Korea). The Livak method was used to measure fold change between the three groups [15].

Statistical analysis
The Kruskal-Wallis test was used to compare VEGF expressions in control, distal and proximal hypospadias' tissues. Post hoc analysis used Mann-Whitney U test for comparison of each group. Values were considered statistically significant with p < 0.05.
The qPCR analyses showed decreasing of VEGF expression in both distal and proximal (− 1.  Table 2).

Discussion
In almost every organ, the biomechanical properties of connective tissues perform essential physiological roles. Biochemical and biophysical properties of ECM are responsible for migration, adhesion, the integrity of individual cells, nutrition and differentiation of cells, angiogenesis, and intracellular contact formation [16]. ECM consists of collagenous (different types of collagen) and non-collagenous proteins, such as fibronectin, elastin, laminin, and other components. Elastin, collagen, and proteoglycans are essential factors in the mechanical properties of tissues [17]. Collagen remains possibly the essential component responsible for preserving the structural stability of the tissue. However, the viscoelasticity of biological tissue does not only involve the amount of components' biomechanical properties. It is the product of a dynamic relationship between cells, angiogenic cytokines, and the ECM, such as VEGF, which maintains the elasticity and integrity of normal tissues [17]. VEGF is a family of closely related growth factors, including several splice variants with numerous biological effects [18]. The production of particular cytokines, including fibroblast growth factor 2 (FGF-2) and VEGF, facilitate cell migration and neovascularization in freshly formed scar tissue [19].
Several studies have proved inelastic dartos tissue in hypospadias. The abnormal findings were found in smooth muscle fiber [20], collagen I and VI [4], total collagen, elastin, and reticulin [6]. Even though dartos tissue has abnormal development of some ECM structure, this study found that dartos tissue has well vascularization.
The present study showed a similar expression of VEGF in patients with hypospadias compared to the control group. VEGF is significantly correlated with the tissue viscoelasticity and the elastic shortening in some organs, such as the cervix. It does not work alone in the elastic mechanism because of crucial tissue mechanical properties such as collagen, elastin, and decorin [9]. A study shows similar blood vessels in the dartos fascia in patients with hypospadias and normal penis [6]. There is strong evidence that this family plays a fundamental role in the growth and differentiation of vascular and lymphatic endothelial cells, but the mechanism remains unclear [9,18].
VEGF is an angiogenic peptide made from macrophages, endothelial cells, and many other cells [21]. It mediates angiogenesis and facilitates the survival of endothelial cells [10]. Integrin β1 acts as a signal transducer to regulate the mechanical environment during this process. Integrin consists of two subunits of transmembrane that modify conformational structures in response to extracellular force [22]. Previous studies identified integrin as a major mechanoreceptor for extracellular signal sensing [23]. Mechanical stimulation enables integrin subunits to be nucleated and clustered to support the maturation of focal adhesions, such as FAK phosphorylation [24].
Pressure overload, particularly integrin β1, can upregulate and activate integrins [25][26][27]. Interestingly, FAK activation was dispensable in angiogenic response mediated by the elastic modulus [9]. Instead, an elevated elastic module upregulated integrin β1 expression in H9c2 cells and activated its Talin-dependent downstream targets, Akt and PI3K. Activating PI3K/ Akt signaling resulted in VEGF being upregulated by its primary transcriptional regulator, HIF-1α [28]. Hypoxia-inducible factor-1 (HIF-1) is a primary transcriptional mediator of the O2 homeostasis hypoxic response. Its master regulator consists of two separate subunits, α, and β [29]. HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions [30]. Hypoxia is the primary stimulus for VEGF release [31]. Hypoxia also reduces the power of the tissue and predisposes it for rupture [9]. Increased VEGF and paracrine are affected by the elevated ECM stiffness in cardiomyocytes. It subsequently stimulates cardiac angiogenesis and the development of VEGF, which can be modulated during cardiac hypertrophy through the elastic modulus of the ECM [8]. Accordingly, Wingate also reported that mesenchymal stem cells in soft VEGF matrices display more mature endothelial cell markers than MSC on soft non-VEGF matrices [32].
However, this study has several limitations, including small sample size and unequal control allocation, a heterogeneous study cohort with both proximal and distal hypospadias. In addition, the tissue studied is the periurethral dartos collected during the excision of the chordee. Most of the distal hypospadias do not require chordee correction or excision. Moreover, VEGF expression needs to be checked in other tissues before a definite conclusion is drawn.  Conclusions VEGF expressions might not affect the severity of hypospadias and chordee, implying the pathogenesis is complex involving many growth factors. Further study with a larger sample size is necessary to clarify and confirm our findings.