Michele Giovinazzo

Bone morphogenic protein-9 stimulates endothelin-1 release from human pulmonary microvascular endothelial cells: A potential mechanism for elevated ET-1 levels in pulmonary arterial hypertension

Abnormalities of signalling for the transforming growth factor beta (TGFβ) family of peptides, including bone morphogenic proteins (BMP), have been described in heritable pulmonary arterial hypertension (PAH). TGFβ can modulate synthesis of the vasoconstrictor and mitogen, endothelin-1 (ET-1), a mediator that contributes to the pathogenesis of PAH. BMP-9 is a circulating peptide recently recognized to affect endothelial function. The stimuli for increased microvascular endothelial production of ET-1 in PAH are unknown. We therefore studied the effects of BMP-9 on ET-1 production by human lung blood microvascular endothelial cells (HMVEC-LBl) in vitro. In vitro, BMP-9 increased ET-1 production by HMVEC-LBl. The effect was identical to TGFβ-1, but BMP-9 and TGFβ-1 combined further increased ET-1 levels by 29%. As compared to TGFβ-1, BMP-9 induced more potent and rapid phosphorylation of Smad 1/5, the downstream signalling molecules of the activin-like kinase 1 (ALK-1) receptor. Moreover, as has been previously shown for endothelial cells of other origin, BMP-9 also induced Smad 2 phosphorylation in HMVEC-LBl. In conclusion, BMP-9 stimulates ET-1 production by HMVEC-LBl in vitro. BMP-9 signals via several Smad pathways. These studies provide novel mechanisms for the potentiation of PAH.

Pulmonary capillary endothelial metabolic dysfunction: Severity in pulmonary arterial hypertension related to connective tissue disease versus idiopathic pulmonary arterial hypertension

OBJECTIVE Pulmonary endothelial dysfunction is intertwined with the development and progression of pulmonary arterial hypertension (PAH). Pulmonary endothelium is an active metabolic tissue in healthy human subjects. This study was undertaken to determine the effects of PAH on pulmonary endothelial angiotensin-converting enzyme (ACE) activity and to identify differences between common PAH types, i.e., PAH related to connective tissue disease (PAH-CTD) versus idiopathic PAH (IPAH). METHODS Nineteen patients with PAH-CTD, 25 patients with IPAH, and 23 control subjects were evaluated. The single-pass transpulmonary percent metabolism (%M) and hydrolysis (both reflecting enzyme activity per capillary) of an ACE synthetic substrate were determined. In addition, the calculated functional capillary surface area (FCSA), normalized to body surface area (BSA), was determined. RESULTS The %M values in patients with PAH-CTD (mean+/-SEM 53.6+/-3.6%) were significantly reduced compared with those in control subjects (P<0.01) and those in patients with IPAH (P<0.03), but were similar between the IPAH and control groups (mean+/-SEM 66.2+/-3.6% and 74.7+/-2.7%, respectively). Substrate hydrolysis was also significantly reduced in patients with PAH-CTD. The FCSA/BSA was significantly reduced in patients with PAH-CTD (mean+/-SEM 1,068+/-118 ml/minute/m2) and in patients with IPAH (1,443+/-186 ml/minute/m2) compared with that in controls (2,948+/-245 ml/minute/m2; P<0.01 for both). At a given cardiac index, the FCSA/BSA tended to be lower in the PAH-CTD group than in the IPAH group. Moreover, unlike in IPAH, a linear relationship between the FCSA/BSA and the diffusing capacity for carbon monoxide (DLCO) was observed in PAH-CTD (r=0.54, P<0.03). CONCLUSION The metabolically functional pulmonary capillary bed appears to be reduced to an equal extent in PAH-CTD and IPAH. However, %M and hydrolysis appear to be reduced in PAH-CTD but not in IPAH, reflecting relatively diminished ACE activity on the pulmonary capillary endothelial cells of patients with PAH-CTD, and showing that pulmonary endothelial metabolic function differs between PAH types. This study also provides the first functional evidence that a reduced DLCO value in patients with PAH-CTD is related to the degree of FCSA loss.

Effects of bone morphogenic proteins and transforming growth factor-beta on In-vitro production of endothelin-1 by human pulmonary microvascular endothelial cells

BACKGROUND Altered endothelial cell (EC)-derived mediator levels, including increased endothelin-1 (ET-1), are hallmarks of human pulmonary arterial hypertension (PAH). Gene mutations for receptors for bone morphogenic proteins (BMP), or transforming growth factor-beta (TGF-beta) cause heritable PAH. The effects of BMPs and TGF-beta on ET-1 production by human pulmonary microvascular EC (HMVEC-LBl) are unknown. METHODS HMVEC-LBl were exposed in-vitro to BMPs 2, 4, and 7 or TGF-beta1 in basal or complete medium. ET production was measured, as well as total cellular protein. Levels of Smad 5 and phosphorylated Smads 1/5 were also measured. RESULTS BMP-4 did not increase ET-1 while BMP-2 increased it minimally in basal medium. BMP-7 increased ET-1, but only at 100 ng/ml. By contrast, TGF-beta increased ET-1 throughout most of the studied dose range. All BMPs and TGF-beta increased levels of phosphorylated Smads 1/5 without depleting levels of Smad 5. CONCLUSIONS With the exception of BMP-7 at high-concentrations, the BMPs that interact with BMP receptor 2, the receptor implicated in heritable PAH, do not or minimally modulate in-vitro constitutive ET-1 production by HMVEC-LBl. TGF-beta increases ET-1 synthesis, and this may have clinical relevance in PAH.

Effects of vascular endothelial growth factor on endothelin-1 production by human lung microvascular endothelial cells in vitro.

AIMS Increased endothelin-1 (ET-1) is a hallmark of pulmonary arterial hypertension (PAH), and contributes to its pathogenesis. The factors controlling ET-1 in PAH are poorly understood. Combined with other stimuli, vascular endothelial growth factor (VEGF) blockade results in PAH-like lesions in animal models, and has been associated with PAH in humans. The effects of VEGF on ET-1 production by human lung blood microvascular endothelial cells (HMVEC-LBl) are unknown. MAIN METHODS We exposed HMVEC-LBl in-vitro to human VEGF-121 (40 ng/mL) in serum-free medium for 7h, in the absence or presence of the VEGF receptor antagonist, SU5416 (3 and 10 μM). ET-1 production was measured in the supernatant. Phosphorylation of VEGF receptor 2 (VEGFR2) was measured by Western blotting after exposure to VEGF without or with SU5416 for 5 and 10 min. KEY FINDINGS VEGF effectively caused VEGFR2 phosphorylation, which was blocked by SU5416. VEGF decreased ET-1 production by at least 29%. In the absence of VEGF, SU5416 increased ET-1 production, by 16% at 10 μM, and SU5416 was able to completely abolish the VEGF effect on ET-1 production. SIGNIFICANCE VEGF may promote vascular health by decreasing ET-1 production in HVMEC-LBl. Blockade of VEGF signaling by SU5416 increases ET-1 levels. The role of VEGF in modulating endothelin production in PAH deserves further study.

ALK2 and BMPR2 knockdown and endothelin-1 production by pulmonary microvascular endothelial cells.

BACKGROUND Many cases of pulmonary arterial hypertension (PAH) are heritable and related to gene mutations in bone morphogenic receptor-2 (BMPR2). These patients consequently may have a signaling imbalance within the transforming growth factor beta (TGFβ) receptor superfamily. The causes of increased endothelin-1 (ET-1), which contributes to PAH, are unknown, and we therefore studied the contribution of various BMPs and their receptors on ET-1 production in vitro, after knockdown of BMPR2 in human pulmonary microvascular endothelial cells (HMVEC-LBl). METHODOLOGY/PRINCIPAL FINDINGS Receptor knockdown in HMVEC-LBl was performed using siRNA to BMPR2, and activin like-kinases 1 and 2 (ALK1, ALK2). ET-1 and TGFβ levels in the medium were measured by ELISA. In some experiments, cells were exposed to TGFβ or BMP7 or FK506 (tacrolimus). Using Western blotting, levels of BMPR2, endothelin ET(B) receptor, phosphorylated SMAD 2 (pSMAD 2), phosphorylated SMAD 1,5 (pSMAD 1,5), ALK1, ALK2, ALK5, TGFβ receptor 2, plasminogen activator inhibitor-1 (PAI-1) and ID1 were measured. BMPR2 knockdown significantly increased ET-1 levels. It did not affect ET(B) receptor or TGFβ levels. TGFβ increased ET-1 levels, with or without BMPR2 knockdown. BMPR2 knockdown did not affect TGFβ (pSMAD 2 and PAI-1) signaling. BMP7 increased ET-1 levels after BMPR2 knockdown but this was prevented by ALK2 knockdown as was the increase in ID1 caused by BMPR2 knockdown. FK506, which interacts with ALK2, increased ET-1 levels and ID1 levels, and this was blocked by ALK2 knockdown. CONCLUSION/SIGNIFICANCE ALK2 may be an important receptor in ET-1 production during BMPR2 knockdown.

Acute Vasodilator Responsiveness and Microvascular Recruitment in Idiopathic Pulmonary Arterial Hypertension

Background: Idiopathic pulmonary arterial hypertension (IPAH) may be characterized according to the hemodynamic response to an acute vasodilator challenge (1). Criteria for response are an acute reduction in the mean pulmonary arterial pressure (mPAP) greater than 10 mm Hg resulting in an mPAP less than 40 mm Hg without a decrease in pulmonary arterial blood flow (that is, cardiac output) (2). Approximately 90% to 95% of patients do not respond and are treated with approved therapies for IPAH; their long-term prognoses vary. The few who do respond generally improve clinically with long-term, high-dose calcium-channel blocker therapy and have an excellent prognosis (3). The lung normally accommodates increased blood flow by recruiting underperfused microvasculature and downstream capillary surface area (CSA) (4). Histologic abnormalities associated with IPAH physically obstruct luminal flow in the precapillary microvessels, reducing perfusion in the distal capillary microvasculature. Therefore, we proposed that nonresponders would be unable to recruit substantial CSA during an acute vasodilator challenge. Conversely, the increased pulmonary vascular resistance (PVR) noted in responders would be based on vascular tone rather than cellular obstruction and CSA would be recruited during a vasodilator challenge. We tested this hypothesis by determining pulmonary endothelial functional CSA (FCSA) with a validated assay based on pulmonary capillary, endothelium-bound, angiotensin-converting enzyme (PCEB-ACE) activity being proportional to perfused pulmonary CSA (58). Methods: Fourteen patients with IPAH were studied after providing informed consent (1, 9). Baseline data for 13 of these patients were previously published in a study involving a larger group in which normal FCSA was also determined (5). None had received IPAH-specific therapy or had saline contrast evidence of intracardiac shunting on echocardiography. The Jewish General Hospital Research Ethics Committee approved the protocol (protocol 00-034). Standardized right heart catheterization was done, and a femoral artery sheath was inserted. After a 30-minute rest, baseline hemodynamic measurements followed by PCEB-ACE metabolic activity were measured before a vasodilator challenge. Next, an acute vasodilator challenge was done using intravenous epoprostenol (Flolan, GlaxoSmithKline Canada), PCEB-ACE metabolic activity and hemodynamics were measured at the peak epoprostenol dose, determined individually by maximum hemodynamic response or adverse effects, and epoprostenol infusion was discontinued. An indicator-dilution method under first-order reaction conditions determined the percentage of single-pass, transpulmonary metabolism and hydrolysis of 3H-Benzoyl-Phe-Ala-Pro (BPAP), a highly specific and hemodynamically inactive substrate for PCEB-ACE (6). Functional CSA was calculated and normalized to body surface area (6). Values among nonresponders before a vasodilator challenge and at the peak epoprostenol dose were compared with paired t tests. Two-tailed Pvalues less than 0.050 were considered significant. Results: Table 1 shows patient characteristics and PCEB-ACE metabolism values and mean and normal values. Patient 7 was the niece of patient 11. In nonresponders, the mPAP did not change with vasodilator challenge. Cardiac output increased, and PVR decreased (P< 0.001). For the 2 responders, the mPAP and PVR decreased and cardiac output increased. Table 1. Patient Characteristics and Mean and Normal Values The mean percentage of single-pass, transpulmonary metabolism and BPAP hydrolysis decreased in nonresponders (P<0.001) but was unchanged in responders. Nonresponders had a low FCSA available for BPAP hydrolysis at baseline compared with normal values (5) that did not increase at peak vasodilator dose. By contrast, the 2 vasodilator responders had higher baseline FCSA, which increased markedly at peak vasodilator dose. Discussion: Most patients with IPAH do not respond to an acute vasodilator challenge. We examined how a lung affected by IPAH handles increased blood flow in relation to capillary recruitment and its physiologic implications. As discussed, the mPAP of nonresponders did not change; however, cardiac output increased, resulting in decreased calculated PVR (2). This effect on pulmonary blood flow allowed examination of whether diseased lungs can accommodate increased flow by recruiting FCSA. The nonresponders had reduced FCSA normalized to body surface area before vasodilator challenge, and an acute vasodilator challenge did not expose more FCSA despite an average 36% increase in cardiac output. These data show the inability of nonresponders with IPAH to open occluded arterioles and thus recruit more downstream capillaries to accommodate the observed increased lung blood volumes that should accompany vasodilator administration. Furthermore, the data provide a mechanistic explanation of the finding that reduction in calculated PVR alone (via increased blood flow) does not imply clinical benefit to the patient (2). The decrease in the percentage of single-pass, transpulmonary metabolism and hydrolysis in nonresponders suggests that the increased blood flow passes through the remaining patent and already maximally recruited vascular tree (10). By contrast, the 2 acute vasodilator responders had a different phenotype with milder baseline hemodynamic abnormalities than most nonresponders (2). Moreover, their baseline FCSA was normal and, most important, increased dramatically during a vasodilator challenge. The absence of a reduction in percentage of single-pass, transpulmonary metabolism and hydrolysis suggests that these patients accommodate the increased cardiac output by true microvascular recruitment and not distention (10). In conclusion, this is the first study to our knowledge to provide evidence that patients with IPAH who do not respond to an acute vasodilator challenge do not recruit FCSA, indicating that any increase in pulmonary blood flow or decrease in calculated PVR does not result in capillary recruitment. By contrast, the few who do respond recruit abundant FCSA, showing that this group can achieve normal levels of lung perfusion. Despite sharing the baseline hemodynamic profile of IPAH, responders and nonresponders represent different vascular phenotypes and perhaps different diseases.

Pulmonary capillary endothelial metabolic function in chronic thromboembolic pulmonary hypertension

Summary.  Background: Chronic thromboembolic pulmonary hypertension (CTEPH) causes physical plugging of large pulmonary arteries as well as a distal micro‐vasculopathy. Pulmonary endothelium is an active metabolic tissue in normal humans. The effects of CTEPH on pulmonary endothelial metabolism are unknown. Objectives: We studied pulmonary capillary endothelium‐bound angiotensin converting enzyme (ACE) activity as an index of endothelial metabolism in patients with CTEPH. Patients/methods: We measured single‐pass transpulmonary per cent metabolism (%M) and hydrolysis of an ACE synthetic substrate and calculated functional capillary surface area (FCSA), normalized to body surface area (BSA), in 13 patients with CTEPH and 23 controls. Results: Mean %M for CTEPH (71.6 ± 4.0% SE) was similar to controls (74.7 ± 2.7%). Substrate hydrolysis (v) was similar for CTEPH (1.47 ± 0.22) and controls (1.51 ± 0.11). However, FCSA/BSA was reduced (P < 0.01) for CTEPH (1530 ± 218 mL min−1*m−2) as compared with controls (2948 ± 245). Conclusions: The metabolically functional pulmonary capillary bed is reduced in CTEPH. However, because %M and hydrolysis are preserved, this points to a reduction in functional capillary surface area rather than reduced ACE activity on the pulmonary capillary endothelial cell. The reduction in functional capillary surface area may just be a result of decreased capillary recruitment because of upstream vascular plugging by chronic organized thrombus.