Журнал «Здоровье ребенка» 6 (49) 2013

Introduction. Investigation and experiments for determination of causes and mechanisms of pulmonary hypertension development have been carried out during the course of the century. At the same time, some aspects of pulmonary hemodynamics in children with broncopulmonary dysplasia (BPD) still remain unclear. Peculiarities of pulmonary circulation in children, who were born at saccular stage, are not determined; possibility of growth and recovery of pulmonary vascular bed in early childhood and predictors of chronic cor pulmonale are under investigation. The article presents peculiarities of embryonic and fetal periods of pulmonary vessels development, intrauterine and extrauterine stages of tone control, etiology and mechanisms of pulmonary hypertension development in children with bronchopulmonary dysplasia and it also provides data conserning authentic investigation of neovascularization, fibrosis and vasodilation factors with BPD. 

It is common knowledge that pulmonary circulation develops along with alveolarization [7]. Vascularization occurs due to integration of endothelium, mesenchyme and epithelium, surrounding bronchial buds, in the condition of low рО₂ (as compared to postnatal period) and depends on extracellular matrix proteins and also on transmitting integrin receptors. Electronic microscopy, carried out at embryonic period, allowed to determine primitive endothelial cells, the precursors of vascular endothelium. Visceral mesodermic cells developed into angioblasts and ranged in extracellular matrix as coordinating cells (cord-like cells). Angioblast was observed to produce first generation of endothelial cells without specificity. Daughter cells of angioblast were found to have features of arterial or venous circulation segment. Сord-like cells formed large vessels through gathering muscular and endothelial cells of the arterial wall and formed capillary network. Apart from vasculogenesis cord-like cells also coordinated alveolar growth [7]. The role of matrix protein, receptors, factors of promotion, differentiation and growth in angiogenesis is currently under investigation.  Thus, the concept of pulmonary circulation in adults with emphasis on angiogenesis peculiarities during intrauterine development is changing. It is still unclear how the cells range and form different functions.

It is known that development of vessels takes place with the beginning of expression by primordial endothelial cells of bronchial buds of fetal liver kinase (flk-1) with activation of VEGF [3]. Cord-like endothelial cells begin to form the tube and surrounding blood islets. As a result it leads to the formation of structure, resembling microvessels. Channelization with vessels leading from venous sinus to capillaries begins in human embryo from the 50^th day of gestation. Primitive capillary networks develop during the course of about 10 weeks and results in the contact between the network and veins located near distal regions of the bronchial buds. This is followed by the formation of vascular network which accompanies alveoli. Thus, vessels develop in a definite order: capillaries –   – pulmonary veins – venous lumen – conjunction of veins with capillaries – channelization of bronchial and pulmonary arteries – their conjunction with capillary network. As a result, at the 19^th week the capillary network becomes part of alveolar wall, forming alveocapillary membrane [1,4]. Matrix capillary provide a connection between mesenchymal, epithelial and endothelial cells. This connection is so strong that pulmonary mesenchyme degenerates without endothelial cells.

When damaged, vascular cells quickly recover and during recovery show anatomically correct growth regardless of mitogen character. Vessel remodeling at embryonic period takes place at the account of muscular cells of a vessel, which are similar to embryonic ones [5]. Growth at the expense of neointima and media is possible only during intrauterine development. After birth it occurs at the account of media. Recruitment of 80% circular pool takes place every 24 hours. After birth replication becomes significantly slower. Basal membrane proteins and matrix metalloproteinases play a major degrading and pro-growth role. It is proved that intrauterine uncoordinated signals can slow down the growth of alveolar space, holding the impairment of permeability of alveolocapillary membrane after birth. Small doses of nicotine during pregnancy induce pulmonary hypoplasia with abnormal development of circulation.

By the end of first half of pregnancy primitive bronchi with vessels and branches of respiratory tract are already formed. From the 18^th to 26^th week of gestation alveolar space is growing exponentially. During fetal period the number of arteriole generations grows (from 5-6 to 40 branches) and pulmonary volume of vessels increases tenfold. Contact of alveoli for the exchange takes place at the 18-20^th week. The authors examined 200 newborns beginning with the 20-40^th week of gestation to determine pulmonary circulation at the last weeks of pregnancy. Thus, ultrasonography showed that the volume of right heart chamber increased by 60% and blood volume by 8%. Pulmonary volume increased quicker than pressure in pulmonary vessels. Prematurely born infants were found to have different structure of supernumenary arterioles: there were 3-4 times as many supernumenary arterioles with a valve and they were more sensitive to NO, 5- hydroxytryptamine, cGMP and less to thromboxane [5].

Supernumenary arteries play an important role in pulmonary circulation, particularly during the last stages of fetal development. Supernumenary arteries branch from bronchial arteries and leave the apex of acinus. Following which they get into parenchyma, disintegrating into fine vessels and open to capillary network of lungs. Supernumenary arteries are the conductors between bronchial epithelium and alveolar septa. For instance, supernumenary artery is an independent conductor from the beginning of acinus.

Growth of vessels depends on normal oxygenation. Growth slows down in low oxygenation. Hypoxic pulmonary vasoconstriction was observed in newborns of the 30-36^th weeks of gestation. Pressure in pulmonary artery is the most evident factor, which controls fetal pulmonary circulation. Low pressure in pulmonary artery is maintained by high arterial tone of pulmonary arteries, necessary during intrauterine development. After birth high arterial pressure decreases due to a decrease in vascular tone. The research of J.  Rasanen provided evidence concerning an increase in blood volume in pulmonary artery in fetus of 31-36 weeks, a decrease in stream through arterial duct and oval window when mother breathes in highly oxygenated mixture [5]. Such changes were not observed in fetuses of 20-26 weeks of gestation. These findings allow to draw the conclusion that fetal oxygen-dependent regulation of pulmonary circulation develops from the 31^st week of gestation. Fetal mechanisms of pulmonary vascular control, such as NO and potassium ion channels also differ from extrauterine ones. High oxygen pressure in fetal lungs activates calcium-sensitive potassium channels (К_Са) and enables the discharge of potassium ions from smooth-muscle cells, which contributes to vasodilation. Though К_Са chаnnels are mediators, vasodilation is promoted by NO, synthesized by mediators of endothelium. During later stages of gestation NO synthesis is provided by smooth muscles (Fig.1). NO is the most powerful vasodilator. Endothelial cells contain enzymes of nitric oxide (eNOS), which catalyze the production by NO endothelial cells, converting amniotic acid, L-arginine (L-arg) into citrulline. NO is produced by endothelial cells and easily passes into media, causing relaxation in the smooth muscles of the vessels. mRNA for eNOS develops in pulmonary arteries of sheep within the last days of the first half of gestation period and plays a major role in fetal pulmonary vasomotor control. Signaling from mRNA to eNOS and activation develop within the last days of pregnancy.  Moreover, eNOS controls normal vascular development [5].

There is evidence of the interrelation between рО₂  and vasodilation through NO. Oxygen activates NO in endothelium, though its effect is short-term. Trials with acute hyperoxia showed a 2-fold increase in blood flow and vessels resistance. However, in two hours the flow and resistance return to previous indices. 

Transient vasodilation is conditioned by acetylcholine. The effect is increased by cGMP (relaxation of smooth muscles of the vessels) and other endothelium-dependent vasodilators, such as histamine, bradykinin, tolazoline. Vasodilators contribute to the closing of arterial duct through activation of endothelial factors. 

Endothelin- 1 is another important vasodilator. Peptide, regulated via Endothelin-A receptors (ЕТа) (vasoconstrictors) or Endothelin-В receptors (ЕТв) (vasodilators). Receptors are located along pulmonary capillaries. Maximum quantity develops by birth. Examination of endothelin receptors in fetuses showed that ETa receptors blocking induces vasodilation and does not produce effect in case of ETв receptors. With reference to these findings researchers suggest that receptor balance at fetal period is aimed at vasoconstriction. ЕТа receptors are determined within early stages, an increase in ЕТв receptors occurs during late stages of gestation. Subjects with pulmonary hypertension were found to have a reduction in ЕТв endothelin receptors after birth. Continuous blocking of ЕТв receptors contributes to an increase in Endothelin-1 level [5].

During the study of pulmonary hypertension development we carried out acute intrauterine compression of arterial duct in sheep. It resulted in an increase in ejection from the right ventricle, an immediate increase in pressure in the right ventricle, an activation of endothelium-dependent mechanisms of vasodilation. These changes continued for about 2 hours. Administration of oxygen in acute compression resulted in a continuous vasodilation. Vasodilation effect was not observed in chronic compression of arterial duct for 9-14 days and further administration of oxygen to mother. Moreover, adequate vasodilation was not observed until birth. Hypoxemia developed in spite of ventilation with 100% provided to mother. Lambs, born following ductal intrauterine compression, were found to have pulmonary hypertension, hypoxia and low blood pressure in the lungs which persisted for several days. Infusion of acetylcholine and endothelium-dependent vasodilators did not provide a sufficient decrease in pulmonary hypertension; resistance of hypoxemia was found to persist. Administration of endothelium-independent vasodilators (natriuretic peptides) and insertion of NO to respiratory tract resulted in dilation of pulmonary artery. This provides evidence of endothelial factors activation without vasodilation and of a major role of endothelium in pulmonary vessels tone. Great capacity for vasoconstriction was also observed after birth, capacity for vasodilation was insufficient. 

Thus, endothelium state is the key to pulmonary hypertension. Fetal arterial wall has a great capacity for replication, which is conditioned by presence of specific isoenzyme of protein kinase С (РКС), which is observed during fetal period and disappears or reduces in adults. Fetal muscular cells and fibroblasts demonstrate a more evident replication as a reaction to hypoxia than cells in adults and vascular wall shows a poorer response to exogenous mitogens. Apoptosis and matrix metabolism are greater in fetal vascular wall and smooth muscle cells are multifunctional. A more evident stimulation of fibronectin, elastin with a continuation of abnormal stimulus during neonatal period and an increased sensitivity to stimulation in early childhood is a peculiarity of matrix intrauterine synthesis. Prematurely born infants develop a more significant vasoconstriction as a reaction to hypoxemia and pulmonary hypertension persists yet for a long period of time after low рО_2. High pressure in pulmonary artery quicker than in adults results in constriction of vascular wall and includes proliferation of fibroblasts, synthesis of connective tissue, activation of cytokines and mitogens, matrix protein and transformation into fibroblasts, which are deposited along the pulmonary artery. Chronic hypoxia contributes to initiation of long-term fibrosis. Besides, any injury of arterial vascular wall subsequently activates discharge of fibroblasts, neomusculinization of vessels and enlargement of media in even greater manner.

The above mentioned data provide evidence for a necessity of carrying out investigation concerning tone in children with bronchopulmonary dysplasia, who were born before the term of gestation and who have factors, contributing to intrauterine inhibition of vascular wall growth and vascular tone regulation mechanisms.

Objectives. Improvement of diagnostics of the causes and mechanisms which lead to the formation of pulmonary hypertension in children with bronchopulmonary dysplasia by studying age-related dynamics of VEGF, TGF-β₁ and cGMP and also their interrelationship with hypoxia and acid-base balance of blood and pressure in pulmonary artery.

Materials and methods. We examined 82 infants at the age of 1-36 months with bronchopulmonary dysplasia (main group), who were treated in the center of diagnostics and treatment of bronchopulmonary dysplasia within the period of 2007-2013. Bronchopulmonary dysplasia was diagnosed according to international classification of diseases of the 10^th edition. The group of comparison comprised 24 infants at the age of 1-36 months, who were born prematurely, had respiratory impairments but did not develop bronchopulmonary dysplasia. The examined infants were born at different terms of gestation. In order to randomize the sample we calculated adjusted age by the formula: А (c) =  – 40 + (А(g) + А (p)) / 4 . Where А(c) – adjusted age, А(g) – gestation age in weeks,  А (p) – passport age in weeks. Oxygen saturation was determined with pulse oximetry, unit «YUTASOKSI-201». Acid-base balance of blood was studied by means of blood gas analyzer АВL-5. Pressure in pulmonary artery was determined by echocardiography with Doppler effect. Growth factors (VEGF and TGF-β₁) were determined by enzyme-linked immunoassay (ELI) of blood serum and cGMP –induced sputum by ELI following induction by 3%NaCl/  Statistical processing of data was performed by non-parametric statistics method: we determined median and quartiles, reliability was assessed by Kolmogorov-Smirnov method, correlation by Spearman method.

Results and their discussion. Children with bronchopulmonary dysplasia were found to have a statistically reliable increase in TGF-β₁ in blood serum in all age groups (р<0,05) (Table 1). This provides evidence to the fact of fibrosis activation and inhibition of pulmonary ontogenesis within the first three years of life without a trend toward a decrease in TGF-β₁ by three years of life. At that, this index showed a slight growth at the third year of life.

We determined a dependency between TGF-β₁ and indices of SatO₂, рСО₂  and pressure in pulmonary artery (Table 2). These data allow us to draw a conclusion concerning a direct reliable dependence of increase pressure in pulmonary artery in fibrosis activation and inhibition of pulmonary ontogenesis. Thus, increased pressure in pulmonary artery can be regarded as a predictor of unfavorable outcome of bronchopulmonary dysplasia with the development of pneumofibrosis. Decreased SatO₂ probably activates fetal muscular cells and fibroblasts, which is proved by a statistically reliable dependence on TGF-β₁ (р<0,05). We consider high activity of TGF-β₁ to be a result of hypoxia, taking into account a possibility of long-term preservation of endothelium trend to fibrosis and vasoconstriction following permanent episodes of hypoxia. These data require further investigation for determination of vasoconstriction preservation terms in children with bronchopulmonary dysplasia following hypoxia and influence on the obstruction of vascular bed of oxygen-dependent and endothelium-dependent mechanisms.

Table 1.

Indices of VEGF and TGF-β₁ general levels in blood serum of infants with bronchopulmonary dysplasia and  examined infants of the comparison group

* – statistical reliability when comparing the indices of the main and comparison groups

Vascular endothelial growth factor VEGF was lower in infants with BPD. Indices reduced evidently from the second year of life in the main group (р<0,05). We regarded the data as inhibition of VEGF activation by сord-like receptors and subsequently growth of vessels within the first three years of life in children with bronchopulmonary dysplasia. Correlation of vascular endothelial growth factor level with the level of pressure in pulmonary artery also determined statistically reliable connections (р<0,05). Thus, increased pressure in pulmonary artery inhibited the growth of pulmonary vessels. Considering data provided by other researchers concerning the predominance of vasoconstriction in prematurely born infants, it is possible to draw a conclusion that a delay in angiogenesis reduced the volume of pulmonary vascular bed, increased pressure in pulmonary vessels and contributed to matrix synthesis impairment.

Table 2.

Correlation connections of VEGF and TGF-β₁ in blood serum and cGMP in induced sputum with pressure in pulmonary artery (р PA), saturation of oxygen (SatO₂) and indices of acid-base balance of blood in infants with BPD (n=82)

The level of cGMP in sputum, which is a powerful vasodilator, was significantly increased in all the age groups (Table 1) and showed the most statistically evident correlation with high pressure in pulmonary artery. In view of the foregoing it is possible to consider hyperactivation of vascular endothelium-dependent factors of vasodilation in response to the elevation of pressure in pulmonary bed in children with bronchopulmonary dysplasia. The absence of a significant lowering of pressure as a response to vasodilators allows to consider incompleteness of vasodilation receptors in this category of children. Hypoxia and hypercapnia also had an impact on the level of cGMP in sputum (р<0,05). At that there was a statistically reliable negative dependency of oxygen on the level of cGMP, which gives grounds for consideration of vasodilation stimulation by means of increased oxygen pressure and vice versa in children with BPD.

Conclusions:  Children with bronchopulmonary dysplasia were found to have a statistically reliable increase in TGF-β₁ (р<0,05) and cGMP (р<0,01-0,001), a reduction in VEGF (р<0,05), which indicates inhibition of angiogenesis, activation of fibrosis and endothelium-dependent factors of vasodilation from the 1^st to 36^th month of adjusted age. We have determined a statistically reliable direct dependence of activation of TGF-β₁ of blood and cGMP of sputum and also an invert correlation of VEGF of blood from рPA. Thus, pulmonary hypertension is an unfavorable factor of fibrosis activation and angiogenesis inhibition in children with BPD. A decrease in saturation and oxygen partial pressure resulted in moderate activation of cGMP, however cGMP in children with BPD did not provide a sufficient pressure reduction in pulmonary artery.