Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient

Abstract

AIM: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients.

METHODS: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis.

RESULTS: Expression of local renin mRNA in the liver of control group was (0.19 ± 0.11), significantly lower than that in splenic artery(0.45 ± 0.17)or splenic vein(0.39 ± 0.12) respectively, (P < 0.05). Expression of local angiotensinogen mRNA in the liver was (0.64 ± 0.21), significantly higher than that in splenic artery(0.41 ± 0.15) or in splenic vein (0.35 ± 0.18) respectively, (P < 0.05). Expression of local renin mRNA in the liver, splenic artery and vein of PH group was (0.78 ± 0.28), (0.86 ± 0.35) and (0.81 ± 0.22) respectively, significantly higher than that in the control group, (P < 0.05). Expression of local angiotensinogen mRNA in the liver, splenic artery and vein of PH group was (0.96 ± 0.25), (0.83 ± 0.18) and (0.79 ± 0.23) respectively, significantly higher than that in the control group, (P < 0.05). There was no significant difference between the liver, splenic artery and vein in the expression of local renin or local angiotensinogen mRNA in PH group, (P < 0.05).

CONCLUSION: In normal subjects the expression of local renin and angiotensinogen mRNA was organ specific, but with increase of the expression of LRAS, the organ-specificity became lost in cirrhotic patients. LRAS may contribute to increased resistance of portal vein with liver and formation of splanchnic vasculopathy.

INTRODUCTION

The initial clues to the presence of an extrarenal or tissue RAS were suggested in the studies of hypertension. Biochemical and histologic evidences have been established for the existence of a tissue-based RAS within a variety of tissues such as blood vessels, liver, kidney, spleen. Many researchers documented that this RAS system was functionally independent of the endocrine system[1-3] and called tissue or local RAS (LRAS). Its activition includes both short and long-term regulatory roles in cardiovascular homeostasis and secondary structural changes, instead of short-term regulatory profile for endocrine RAS. Locally generated AngII plays a significant role not only in controlling the growth of vascular smooth muscle cells (VSMC), hepatocytes and hepatic satellite cells (HSC was one of the most important cells in liver fibrosis[4-6]), but also in regulating local vascular tone including hepatic microcirculation. Hyperdynamic circulation and splanchnic vasculopathy were the common pathological process and changes in cirrhotic portal hypertension[7-9], vascular and hepatic RAS may have great contribution to it. Through detection of expression of the two components of LRAS, the relationship between local renin and angiotensinogen and cirrhotic portal hypertensions was investigated.

MATERIALS AND METHODS

Materials

The liver tissues and a section of splenic artery and vein were obtained during the operation of esophagogastric devascularization and splenectomy in 20 cirrhotic portal hypertensive patients. The same samples were obtained during splenectomy and partial hepatectomy in 24 controls.

13 male and 7 female patients were included in this study with mean age of 49 ± 21, mild or severe gastroesophageal varices and splenomagely were found in all patients.

16 male and 8 female subjects with mean age of 39 ± 17 served as control, 12 of them underwent partial hepatectomy for hepatic trauma and 12 underwent splenectomy for splenic injury. Those with hepatitis or hypertension were excluded.

A portion of the resected tissues was routinely fixed with 10% formalin and embedded in paraffin and cut into sections, another portion of tissues was stored in liquid nitrogens at -80 °C for use.

Methods

Immunohistochemical analysis To investigate splanchnic vascular changes in these patients, we took the splenic veins by using monoclonal anti-vascular smooth muscle cell α-cm-actin antibody, PCNA, and the splenic veins were stained with HE. Immunohistochemical analysis was performed according to routine methods as suggested by the manufacturer.

Preparation of specimens for electron microscopic observation Fresh vascular tissues were made into cubes of 1 mm³ and prefixed for 2 h in 2.5% glutaraldehyde, and then postfixed at 4 °C for 2 h in 1% osmic acid. Alcohol of increasing concentrations and acetone were used for dehydration. The specimens were then embedded in epoxy resin EPON 812 and cut into ultrathin sections. Plumbum-double-staining was used to prepare the samples for ultrastructural observation under transmission electron microscope (Opton EM 10C Model).

Reverse-transcription polymerse chain reaction (RT-PCR) Messenger RNA (mRNA) levels of local renin and angiotensinogen were assessed by RT-PCR analysis using β-actin as the house keeping gene. Total RNA was prepared from frozen tissues of the liver, splenic artery and vein. Diluted complementary DNA was cloned in a total volume of 50 μL containing DEPC 31.5 μL, buffer solution 5 μL, dNTP 4 μL, 2.5 mmol/L MgCl₂ 3 μL, primers 20 μmol*4 μL, cDNA 2.5 μL, and TAG 0.25 μL. The PCR conditions were at 94 °C for 4 min followed by 35 cycles at 90 °C for 30 sec, at 54 °C for 30 sec, and at 72 °C for 1 min. Total RNA was also analyzed for β-actin transcript expression and PCR for 28 cycles for β-actin. (Table 1).

Table 1.

Primer sequences

MRNA	Sequence	Size of products
Renin sense	5'-TCTCAGCCAGGACATCATCA-3'	288 bp
antisense	5'-AGTGGAAATTCCCTTCGTAA-3
Angiotensinogen		256 bp
sense	5'-TGTTGCTGCTGAGAAGATTG-3'
antisense	5'-CCGAGAAGTTGTCCTGGATG-3'

RT-PCR products were visualized under ultraviolet and analyzed by computer which provided the data for analysis. Study values were normalized as a ratio to the β-actin signal in each sample, and each value was analyzed as a transcriptional index (transcript of the interest mRNA expression in samples of interest/β-actin mRNA expression in samples of interest).

Statistical analysis

P value < 0.05 was considered statistically significant, and all variable data were summarized in terms of mean ± SD and analyzed by Student t test using SAS software.

RESULTS

Immunohistochemical staining

In these patients, such typical cirrhotic changes as hepatocyte degeneration, necrosis, pseudolobule formation and fibrosis were seen, and splenic vein wall was thickened and VSMC in media tunica was disorderly arranged (Figure 1, Figure 2). With α-cm-actin antibody staining method, VSMC could be seen in the intima of splenic vein, suggesting migration of VSMC from media to intima. Hyperplasia of VSMC could be seen in both splenic artery and vein by PCNA staining (Figure 3, Figure 4), there was no obviously positive staining in the control group.

Figure 1.

The liver tissue in portal hypertensive patients with HE staining (× 400).

Figure 2.

Splenic vein in portal hypertensive patients with HE staining method (× 400).

Figure 3.

Splenic vein in portal hypertensive patients by immunohistochemical staining with α-cm-actin antibody (× 400).

Figure 4.

Splenic vein in portal hypertensive patients by immunohistochemical staining with PCNA (× 400).

Electron microscopic observation

In the splenic vein of these patients, endothelium was damaged with adhered thrombus, VSMC of media migrated into the subintima under transmission electron microscopy (Figure 5, Figure 6).

Figure 5.

Splenic vein of portal hypertensive patients under transmission electron microscope (× 4000).

Figure 6.

Splenic vein of portal hypertensive patients under transmission electron microscope (× 6000).

RT-PCR analysis

In the control group, the expression of local renin mRNA in the liver was significantly lower than that in the splenic artery or vein, P < 0.05. Expression of local angiotensinogen mRNA in the liver was significantly higher than that in the splenic artery and vein, P < 0.05. There was no significant difference in the expression of local renin or angiotensinogen mRNA between splenic artery and vein. (P > 0.05, Figure 7, Figure 8, Figure 9).

Figure 7.

Expression of human β-actin mRNA (Note: from left to right there was PCR marker and lanes 1-6, figures 8, 9 were the same).

Figure 8.

Expression of local renin mRNA in the liver, splenic artery and vein from the controls and cirrhotic portal hypertensive patients. Lanes 1, 3, 5 were splenic vein, artery and liver from controls, Lanes 2, 4, 6 were from patients.

Figure 9.

Expression of local angiotensinogen mRNA in the liver, splenic artery and vein from controls and portal hypertensive patients. Lanes 1, 2, 3 were splenic vein, artery and liver from controls, Lanes 4, 5, 6 were from portal hypertensive patients.

In cirrhotic portal hypertensive patient group, expression of local renin and angiotensinogen in liver, splenic artery and vein were all significantly higher than those in the controls respectively, P < 0.05. Expression of local renin and angiotensinogen mRNA in the liver was not significantly different from those in the splenic artery or vein in pH group, P > 0.05. The concrete data are listed in Table 2.

Table 2.

Expression of local renin (Lr) and angiotensinogen(Lan) mRNA in the patient and control groups

	Control group (n = 12)			Patient group (n = 20)
	Liver	Splenic artery	Splenic vein	Liver	Splenic artery	Splenic vein
Lr	0.19 ± 0.11a	0.45 ± 0.17	0.39 ± 0.12	0.78 ± 0.28b	0.86 ± 0.35b	0.81 ± 0.22b
Lan	0.64 ± 0.21a	0.41 ± 0.15	0.35 ± 0.18	0.96 ± 0.25b	0.83 ± 0.18b	0.79 ± 0.23b

The data were expressed as mean ± SD. Lr and Lan mRNA in the patient group versus those in the control group,

^bP < 0.05.Within the same group, Lr and Lan mRNA in the liver versus those in splenic artery or vein respectively,

^aP < 0.05.

DISCUSSION

The results demonstrated that expression of hepatic renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients was significantly higher than that of the controls. Therefore the end product of LRAS, the synthesized local AngII increased and exerted a strong effect on hepatic microcirculation via its paracrine pathway.

Firstly, local AngII constricted blood vessels as well as sinusoids leading to increase of intrahepatic portal venous pressure through an increase in intracellular calcium in VSMC. Although VSMC was absent in the hepatic sinusoid, hepatic stellate cells (HSCs) expressed receptors for AngII, could also contract and increase the intrahepatic resistance[10-12].

Secondly, AngII has been shown to induce cell proliferation in various cell types, including hepatocytes and HSCs and was considered as a mediator of hepatic fibrogenesis[3,13]. Local angiotensinogen also induced increase of TGF-β mRNA expression which was an important growth promoting factor for HSC[14,15]. It was demonstrated that AngII could be a mitogenic factor for activated human HSCs through MAPK-dependent pathway[3,16]. Significant relationship was seen between high TGF-β and AngII production and the development of progressive hepatic fibrosis caused by hepatitis C virus[17,18]. AngII was also involved in the development of fibrosis in the heart and kidney through enhancement of TGF-β production[19]. In vitro study found that AngII could increase mRNA levels for collagen types I and III, procollagenα(I) and fibronectin in cardiac fibrosis[20]. Therefore it is possible that hepatic RAS plays an important role in the collagen synthesis, hepatic fibrosis and cirrhosis. In this study, the cirrhotic portal hypertension induced hepatic RAS activity which increased and accelerated the cirrhotic process and portal hypertension. By interrupting the vicious cycle, it was possible for medical treatment to prevent further progression of the disease. It has already been confirmed that catoprill could reduce the expression of procollagenI significantly and prevent liver fibrogenesis in a rat model of hepatic fibrosis[21-23].

The experimental data illustrated that expression of local renin and angiotensinogen mRNA in the splenic artery and vein of cirrhotic portal hypertensive patients was significantly higher than that of the controls and suggested that portal hypertension led to activation of LRAS of splenic vessels. The mechanism probably might be as follows: (1) Endothelial injury was caused by splanchnic hyperdynamic circulation and high blood flow shear, and the endothelial cells were the key site of LRAS metabolism[2]. (2) The splenic vessel wall was stretched by the increment of splanchnic blood flow. (3) Influence of other vasoactive substance.

When the expression of LRAS increased in splenic vessel, it could participate in many pathologic processes. Firstly, vascular RAS induced VSMC proliferation and enhanced progression of vascular remodeling. AngII induced hypertrophy, proliferation and migration of VSMC[24-26] with modulation of expression of C-fos[22], C-jun, C-myc and synthesis of cytokines such as PDGF, b-FGf [23] etc. In this study, changes of VSMC in splenic vein of PH patients could be seen by HE stain, immunohistochemistry and electron microscopy. These suggested that vascular RAS was closely related with the structural alterations of the splenic vein. Response of VSMC to hypertension and injury of blood flow shear included: hypertrophy, proliferation and remodeling. As a result, the vascular RAS played an even more important role than endocrine RAS[27-30]. In addition, the matrix modulation was another key event in remodeling and vasculopathy[7,8]. Other studies reported that vascular RAS modulated the synthesis of vessel matrix via its effect on expression of PDGF and TGF-β etc[31,32]. Therefore, LRAS plays an important role in vascular remodeling and vasculopathy in portal hypertension.

Secondly, LRAS has vasoconstrictive functions. The response of splanchnic blood vessel to AngII decreased in advanced portal hypertension[33], it was due to decrease of AngII receptor on the blood vessel wall or due to the existence of post receptor dysfunction[34,35]. Furthermore, glucagon and other vasoactive substances could influence the effect of AngII on splanchnic vessel[36,37].

Thirdly, the imbalance of vasoconstrictors and vasodilators was existed in portal hypertension. Studies on vasculature showed that vascular RAS could change the response of blood vessel to other vasoactive substances[29] and vice versa[38]. For example, in the rabbit model of portal hypertension, the response degree of splanchnic vessel to AngII was improved by CO[33], the presence of ET could induce the local renin activity and increase sythesis of AngIIin rat mesentery artery[39], and LRAS could decrease the degradation of endogenous bradykin[30]. All the changes mentioned above could result in imbalance of vasodilators and vasoconstrictors. In this study, local renin and angiotensinogen mRNA expression in the liver was not significantly different from that in the splenic artery and vein in portal hypertensive patients, suggesting that the loss of expression of organ- specificity of LRAS components might be due to disordered metabolism of vasoactive substances.

In conclusion, increased LRAS activity in the hepatic and splenic vessels is due to cirrhotic portal hypertension, and the synthesis of local AngII increases, which contract the hepatic sinusoid, stimulate hyperplasia of HSC and proliferation of VSMC, and also interfere with the metabolism of other vasoactive substances. All these enhance the degree of cirrhosis and portal hypertension. By interruption of this vicious cycle, medical treatment may be able to improve the hemodynamic disturbance and ameliorate the splanchnic vasculopathy and to offer a new way for preventing the complications of portal hypertension[40].