Hypertension is a disease with a high morbidity, disability and mortality rate. There are currently more than 100 million patients in China and more than 50 million patients in the United States. Hypertension is a common cause of coronary heart disease, myocardial infarction, cerebral hemorrhage and cerebral embolism. The pathogenesis of hypertension is complex. In 1997, the Sixth Report of the Joint National Commission on the Prevention, Detection, Evaluation and Treatment of Hypertension showed that only 27.4% of patients treated for hypertension had their blood pressure controlled. In addition, these drugs have short duration of action (no more than 24h), high toxic side effects (hyperkalemia, hypotension, cough and irreversible kidney damage) and need to be taken for life. Since traditional hypertension treatment drugs cannot prevent the occurrence of hypertension and reduce the incidence of hypertension, it is necessary and urgent to seek new hypertension treatment methods and strategies. With the development of molecular biology technology, gene therapy for hypertension has naturally become a hot spot for current research. Gene therapy for hypertension includes both righteous (gene transfer) and antisense (gene suppression) approaches.
1.Righteous gene therapy for hypertension
Justified gene therapy for hypertension refers to the use of liposomes, adenovirus or retrovirus as vectors to transfect target genes into the body by intravenous injection or local injection into target tissues to express the corresponding proteins for the purpose of treating hypertension.
(1) Adrenomedullin gene
Adrenomedullin (ADM) is a peptide isolated and purified from the extract of pheochromocytoma tissues by Japanese scholars. It is composed of 52 amino acids and has the functions of vasodilator, sodium excretion and cardiotonic in vivo. Adrenomedullin (ADM) is expressed in cardiac myocytes, vascularsmoothmusclecell (VSMC) and endothelial cells, and ADM has effects on vasodilatation, inhibition of VSMC proliferation and migration, increase of renal blood flow and promotion of sodium excretion, etc. Chao et al. The results showed that after a single injection of ADM plasmid, ADM was stably expressed in kidney, heart and lung tissues and caused a significant decrease in blood pressure for more than 5 weeks; after the first transfection for 5 weeks and an additional injection, the antihypertensive effect could be maintained for 3 weeks and blood pressure could be decreased by up to 2.93 This experiment suggests that transfection of human ADM gene can lower blood pressure for a long time.
(2) Atrial natriuretic peptide gene
Atrial natriuretic peptides (ANP) are a family of active peptides discovered in the 1980s, the most important of which is a 28-amino acid peptide in humans. The effect of reducing BP is achieved through the mechanisms of diastole, reducing peripheral resistance, increasing glomerular filtration rate, inhibiting the release of renin, and causing an obvious increase in sodium and urine volume. The atrialnatriureticpeptide (ANP) family consists of three members, including cardiac natriuretic peptide, cerebral natriuretic peptide and C-type ANP, which is a cyclic structure consisting of 17 amino acids. The gene was transfected with adenoviral vector in hypertensive rats (DSS) induced by high salt diet, and after a single intravenous injection of ANP gene for 3 d, it caused a decrease in blood pressure for more than 5 weeks, with a maximum decrease of 4.37 kPa. Morphological tests also revealed that cardiac hypertrophy, glomerulosclerosis, tubular and arterial thickening were significantly reduced, indicating that transfection with ANP gene has a better therapeutic effect on hypertension.
(3) Nitric oxide synthase gene
NO produced by endothelial cells has a role in blood pressure, blood flow regulation, inhibition of platelet aggregation, and inhibition of endothelial cell and VSMC proliferation. The nitric oxide (NO) gene was transfected into SHR by tail vein injection with cytomegalovirus, and a single injection caused a significant decrease in blood pressure that lasted for 5-6 weeks. Other indexes showed that cGMP levels in urine and arteries, nitrite/nitrate levels in urine and serum were significantly increased after NOS gene transfection. However, there were no significant changes in body weight, heart rate, water intake, diet and urine output. This suggests that NOS gene transfection may have a promising application in the treatment of hypertension and cardiovascular diseases [7]. In addition, Alexander et al. experimentally confirmed that human eNOS gene injected through carotid artery using adenovirus as a vector improved NO bioavailability defects and restored endothelial function of carotid artery in rats with primary hypertensive stroke tendency, while superoxide dismutase (Cu-Zsuperox-idedismutase,Cu-ZnSOD) had no such effect. Thus, it provides a new avenue for the study of gene therapy for essential hypertension and renal vascular diseases.
(4) Heme oxygenase gene
Sabaawy et al. injected retroviruses containing human HO1 cDNA into 5-d-old SHR myocardium in a single session and found that rats expressed HO1 and reduced blood pressure, and that HO1 gene expression showed a decrease in urine output and a decrease in response to elevated intra-arterial pressure. reduced responsiveness, etc. However, it is noteworthy that transfection of HO1 gene reduced blood pressure in rats accompanied by significant body growth.
(5Kinin-releasing enzyme gene)
Kinin-releasing enzyme (kallikrein) is a class of proteases in the body that breaks down certain protein substrates, kininogen, into bradykinin. Bradykinin has vasodilatory activity and is involved in the regulation of blood pressure and local tissue blood flow. In human and animal experiments, bradykinin is one of the most potent diastatic substances known to date. The maximum BP reduction in rats injected with human tissue kinase gene was up to 50 mmHg, and the minimum BP reduction was up to 32 mmHg, which lasted for the whole experimental period. In addition, DOCA-salt hypertensive rats showed a decrease in urine volume, urine protein level and body weight after transfection with human tissue kinase gene. The renal morphological examination showed a significant reduction in glomerulosclerosis, tubular dilatation, and protein tubular pattern.Costanza et al. used adenovirus as a vector to transfect spontaneouslyhypertensiverats (SHR) with human tissue kinin releasing enzyme gene after intramuscular injection, and the results showed that human tissue kinin releasing enzyme gene could promote spontaneous compensatory angiogenesis in animals with normal BP and, of particular importance, corrected the defective vascular regeneration in SHR. Renin-binding protein gene and other genes have also been effective in experimental treatment of hypertension, indicating that righteous gene therapy for hypertension is a promising therapeutic approach.
2. Antisense gene therapy for hypertension
Antisense gene therapy for hypertension refers to the use of liposomes, adenoviruses or retroviruses as vectors to transfect ASODN that can bind to target genes into the body by intravenous injection or local injection into target tissues to inhibit the expression of specific proteins by using the corresponding antisense oligodeoxynucleotides (AS-ODN) or recombinant on The antisense repression or closure of over-expressed genes that cause vasoconstriction and hypertension with the corresponding antisense oligodeoxynucleotides (AS-ODN) or antisense nuclear fragments (e.g., AS-cDNA) recombinant in the expression vector inhibits replication, transcription, post-transcriptional mRNA processing transport and translation processes, thereby suppressing the production of active proteins that cause hypertension (e.g., angiotensin II [Ang II]), lowering blood pressure and reversing The current work on blocking the production of vasoconstrictor substances has been carried out in a number of ways. The current target genes for gene therapy in blocking the production of constricting substances are mainly directed at certain components of the RAS system, such as the AGT and AT1 genes. Clinical trials have demonstrated that RAS inhibition is an important way to treat hypertension, and four antisense components have been used in therapeutic studies for hypertension: antisense AT1R (AS-AT1), antisense AGT (AS-AGT), antisense angiotensin-converting enzymeanti-sense (angiotensin II-convertingenzymeanti-sense,AS- ACE) and antisense β1-adrenergic receptorant (batal-adrenergicre-ceptorantisense,AS-β1).
(1) Type I angiotensin II receptor gene
Gyurko et al. successfully lowered blood pressure in SHR with angiotensin II receptor subtype 1 (AR1R). This was done by injecting antisense oligonucleotides of anti-AT-1 receptor mRNA into the ventricles of SHR, and then measuring the number of AT-1 receptors and angiotensin II receptor subtype 2 (AT-2) receptors of and monitoring the blood pressure of rats. The results showed that the number of AT-receptors in the subthalamic tissue blocks of SHR was reduced by 20%-30%, and the blood pressure of SHR was significantly reduced by up to 49 mmHg. These results provide a reliable basis for anti-angiotensin II receptor gene therapy for hypertension, and Pachori et al. transfected SHR with ASODN of AT1R by retrovirus, which resulted in a sustained decrease in blood pressure in rats, indicating that antisense gene therapy for hypertension is feasible in theory and practice. In addition, an animal model of hypertension and its associated myocardial hypertrophy was established with transrenin rats, and ASODN of AT1R was injected into the myocardium of neonatal rats by retrovirus at once. hypertrophy in the control rats was already very serious at 16 d, indicating that local tissue transfection with ASODN could produce a more desirable therapeutic effect.
(2) Tyrosine hydroxylase gene
Kumai et al. reported that intravenous injection of ASODN with tyrosine hydroxylase (TH) gene could significantly reduce blood pressure, adrenaline/norepinephrine level, TH enzyme activity and TH protein level in SHR, while intravenous injection of ASODN to WistarKyoto rats had no significant effect on blood pressure, and it could significantly reduce catecholamine level, TH enzyme activity and TH protein level. It significantly reduced catecholamine levels, TH enzyme activity and TH protein levels, indicating that systemic application of ASODN inhibiting TH gene was effective in the antihypertensive treatment of SHR.
(3) Angiotensinogen gene
Among the candidate genes for hypertension (EH) studied so far, angiotensinogen (AGT) gene is considered to be the most likely EH-related gene. The human AGT gene is located on chromosome 1q42–43. Applying linkage analysis to affected sibling pairs revealed a significant linkage between the AGT gene and EH, with most reports confirming that the M235T variant of the gene is associated with hypertension, with plasma AGT levels in the order TT>TM>MM genotype. The last study showed that systolic and diastolic blood pressure and plasma AGT concentrations were significantly higher in TT and TM genotypes than in MM patients, and in addition, the proportion of the former with antihypertensive drugs and the proportion with more than two antihypertensive drugs were significantly higher than the latter. It has also been reported that patients with T235 genotype hypertension are more sensitive to the antihypertensive response to angiotensin-converting enzyme inhibitors (AGEI). The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and in the development of hypertension, and AGT is the only substrate in the RAS that produces angiotensin I (AngI). The result was a temporary decrease in plasma AGT levels in SHR, accompanied by a decrease in AGT mRNA in SHR liver, a decrease in plasma Ang II concentration and a temporary decrease in blood pressure. After the injection of antisense ODNs, the blood pressure of SHR was lowered from 178 mmHg to 154 mmHg maintained for about 1 week. The hypotensive effect of antisense ODNs cannot be sustained, and how to prolong the duration of hypotensive effect is also the main direction of future research.
AGT, as an important compound of RAS, plays an important role in the occurrence and development of hypertension in humans and animals, and data show that systemic application of ASODN with AGT gene can reduce SHR blood pressure level. kimura et al. injected AGT gene carried by recombinant adenovirus (recombinationadenoassociatedvirus,rAAV) ASODN was injected into the myocardium of 5-d-old SHR at once, and the onset of hypertension was delayed by 91 d. The rats showed a significant reduction in blood pressure for 6 months after adulthood. The maximum reduction of blood pressure was 3.07 kPa, and the transfected ASODN worked stably in the liver, kidney and heart, and significantly reduced left ventricular myocardial hypertrophy and AGT level in the liver, and no hepatotoxic effects were observed. It indicates that ASODN transfected with rAAV for AGT gene can safely, stably and long-lastingly treat hypertension.
(4) β1-adrenergic receptor gene
However, they have many side effects because they affect the central nervous system and β2 adrenergic receptor (β2AR). Zhang et al. designed an ASODN that specifically inhibits β1 adrenergic receptor (β1AR) mRNA to overcome the shortcomings of traditional β-blockers and produce long-term antihypertensive effects. The results showed that a single intravenous injection of ASODN with cationic liposome carrier could significantly reduce myocardial β1AR density (30%–50% for 18 d), but had no effect on β2AR, and the maximum decrease in SHR blood pressure was 5.07 kPa for 20 d. There was no significant decrease in heart rate, and no significant decrease in radioautography. There was no significant change in β1AR in brain tissue, while it was significantly reduced in heart and kidney tissues (P<0.05). The hypotensive effect of the simultaneously observed β-blocker atenolol on SHR was maintained for only 10 h, and it also induced bradycardia. The results showed that ASODN, which inhibited β1AR mRNA expression, lowered blood pressure longer and more consistently than conventional β-blockers, without significant effects on heart rate, β2AR, or the central nervous system.
(5) Angiotensin-converting enzyme gene
The important role of ACE in RAS and hypertension has been confirmed by numerous studies, and ACE inhibitors are a class of hypertension therapeutic drugs commonly used in clinical practice. Wang et al. focused on the following two questions: (1) whether ASODN inhibiting ACE mRNA can prevent the development of hypertension in juvenile SHR; (2) transfection of ASODN with retroviral vectors in spontaneously hypertensive biparental rats with a retroviral vector to observe whether the anti-hypertensive phenotype was inherited to the offspring. The results showed that myocardial injection of the ACE gene ASODN with retroviral vector in 5-d-old SHR could moderately lower blood pressure for a long time, by (1.12±0.20) kPa, and prevent the development of cardiovascular and renal vascular pathological changes, and reduce ventricular hypertrophy, restore renal artery blood pressure, receptor-dependent contractile response and endothelial cell function; while the blood pressure of WistarKgoto rats transfected with the same gene WistarKgoto rats transfected with the same gene showed no significant changes in blood pressure, suggesting that RAS plays an important role in the pathogenesis of hypertension and that inhibition of this system at the genetic level may be an effective method for long-term control of hypertension.
A unique finding of this study was that spontaneously hypertensive biparental rats transfected with the ACE gene ASODN were able to pass on their acquired antihypertensive phenotype to their offspring, who had the same reduced blood pressure, reduced myocardial hypertrophy, normal renal artery excitation coupling, and normal renal artery endothelial cell function as their parents. The results of Southern blotting and polymerase chain reaction (PCR) showed that the gene ASODN was inserted into the parental genome of SHR and inherited to the offspring. This may be an important reason why the offspring SHR acquired the anti-hypertensive phenotype, and this finding provides a new idea for the prevention and control of human hypertension with a family genetic predisposition.
(5) Other target genes
Some other genes, such as thyrotropinreleasinghormone (TRH), angiotensingeneactivatingelements, carboxypeptidaseY, and cfos of ASODN also has effects such as lowering blood pressure and/or inhibiting VSMC proliferation.
Different antisense components have different biological effects and antihypertensive effects, and the same antisense components have different antihypertensive effects and improvements in pathophysiological and morphological changes depending on the presence or absence of carriers and the type of carriers or the route of entry into the body. However, the vast majority of reports in the literature have shown the superiority of antisense gene therapy: a single injection of antisense components can lower blood pressure or delay the onset of hypertension in the long term and prevent or reverse the pathophysiological and morphological changes associated with hypertension with high selectivity and without the side effects of conventional drugs. The available data suggest that antisense gene therapy is an effective method to control human hypertension in the future.
3. Problems and Prospects
The large amount of experimental data accumulated on nonsense and antisense gene therapy for hypertension fully demonstrates that gene therapy can not only reduce blood pressure consistently and steadily, but also may control the occurrence of hypertension fundamentally and control the family genetic tendency of hypertension, which is beyond the capability of all current hypertension treatment drugs. A large number of experimental data show that gene therapy for hypertension has encouraging prospects, but at present, gene therapy for hypertension is still in the preclinical animal experimental stage, and there are still many problems that we need to overcome and solve, the main problems at present are.
(1) The selection of ideal target genes is the prerequisite for the treatment of hypertension. Only with a thorough understanding of the molecular biology mechanism of hypertension and a clear understanding of the key genes and related genes of pathogenesis is it possible to diagnose and treat hypertension at the genetic level, and hypertension is a multigene abnormal disease, which brings certain difficulties to the definition and selection of target genes, therefore, the selection of multiple targets (combined targets or network Therefore, the selection of multiple targets (combination targets or network targets) for comprehensive treatment should be an important issue in the research of hypertension gene therapy;
(2) Vector construction. The construction of safe, efficient and low immunogenicity vectors is a hot topic in gene therapy.
(3) The transfer efficiency and targeting of gene transfer systems (such as adenovirus, retrovirus, liposome, etc.) need to be further improved, and the long-term effects of transfer systems on human body need to be further clarified.
In general, direct gene transfer methods are preferred over indirect methods. With the development of molecular biology and molecular pathology theories and technologies, and the continuous improvement of the overall level of gene therapy, we believe that gene therapy will become one of the best and effective ways to treat hypertension.