Camouflaged angiogenic BMP-2 functions exposed by pico-paracrine biohybrids

The constant release of human bone morphogenetic protein 2 (rhBMP-2) in the picomolar range (Pico-Stat) from PDLLA-biohybrids led to the detection of intrinsic novel pro- and anti-angiogenic functions of this cytokine. As integrant part in this perspective of previous work, first evidence for the binding of rhBMP-2, as an inverse agonist, to allosteric angiogenic receptors in cocultures of human endothelial cells is reported.

. This protein, rhBMP-2 CHO , is produced by a genetically engineered Chinese hamster ovary cell line, as a disulfide-linked heterodimeric protein mixture of two different polypeptide chains of 114 and 131 amino acids.Each chain is glycosylated at one site with high-mannose-type glycans (Food and Drug Administration, 2004; Committee for Medicinal Products for Human use, 2014; Kenley et al., 1993).As applied, BMP-2 CHO comprises a microheterogenous mixture of three different BMP-2 isoforms and up to 15 additional different glycoforms (Kenley et al., 1993).
Cocultures consisted of endothelial cells (130.000/well) and primary osteoblasts (20.000/well).Relative gene expressions (RQ) of mRNA for various proteins are described in (Dohle et al., 2021).In dose-response experiments, rhBMP-2 COL was added in defined concentrations to the OEC/pOB co-culture system and harvested after 7 days of incubation for fluorescence microcopy and quantitative Nikon NIS image processing as described (Dohle et al., 2021).The pixel values were converted to length according to 1 pixel = 0.46 µm.Cell viability was tested by the LDH cytotoxicity assay CyQUANT ™ as described (Dohle et al., 2021).In desorption experiments, 1 cm 2 (1 mg) fleeces with an rhBMP-2 COL loads of ~6.98 mg/g PDLLA were placed in 1.5 mL flow-through chambers (Sänger et al., 2014).They were perfused with sterile phosphate-buffered saline (PBS) pH 7.4 at a flow rate of 10 mL/h in a 16-channel Watson-Marlow peristaltic pump for 22 days at ca. 22 °C-26 °C.Statistical calculations were done with the PC program GraphPad Prism 4/5 (La Jolla, CA).Prism software fails in exponential decays with r 2 -values of <0.2 when decays parallel the abscissa at t 1/2 > 100-200 days (slope ~zero).Kinetic and thermodynamic constants under non-standard conditions are termed "apparent" (K′, k′).Equilibrium dissociation constants (K D , K 0.5 ) represent affinity constants (Landry et al., 2015).
In contrast to the pro-angiogenic activities of rhBMP-2 COL , the glycosylated species rhBMP-2 CHO was anti-angiogenic, fully inhibiting control angiogenesis (Dohle et al., 2021) and forming instead a cobblestone layer of endothelial cells [not shown, see (Dohle et al., 2021)].
These results were confirmed in Table 3.At a low rhBMP-2 COL , adsorbate loads of 0.33 mg/g and a total length of microvessels of 23.9 ± 5.1 mm formed without an increased expression of VEGF-A (Dohle et al., 2021).At high adsorbate loads of 2.6 mg/g, the total length decreased 5-fold to 4.7 ± 0.44 mm, and in controls lacking adsorbate down 10-fold to intrinsic 2.4 ± 0.046 mm.The knots in the microvessel mesh paralleled the length changes.At low concentrations of rhBMP-2 COL , they totaled a high of 51.9 ± 11.5 knots, decreasing 4.3-fold to 12.2 ± 0.6 at higher concentrations and 12-fold less in controls of 4.3 ± 1.1 knots.These changes were statistically significant (Table 3).
The data of Table 4 are plotted in Figure 3 down to concentrations of 10 −16 M for a classical receptor type.Inhibition increases with the concentration of rhBMP-2 COL .The apparent half-saturation constant is not a classical affinity constant but an "autoinhibition" constant (K′ I 0.5 ) with a value of 3.2 pM.The hypothetical (see dotted line) dissociation constant (K′ D ) for the presumed association of rhBMP-2 COL to endothelial cell receptors lies in the subpicomolar range ~10 −14 M. The anti-angiogenic regulation by rhBMP-2 COL prevents too many blood vessels from being formed as a "high concentration cut-off regulation", in which an apparent agonist turns into an antagonist at high concentrations.
Figure 4 shows a compound extended dose-response plot comprising the data of Table 4 on inverse related angioinduction (Dohle et al., 2021) together with data on direct related osteoinduction [derived from activity measurements with MC3T3-E1 cells in culture (Laub et al., 2007)] and their respective affinity constants.The inverse angiogenic branch saturates at ca. 15 mm and the direct osteogenic branch at 1.2 OD units.The apparent halfsaturation autoinhibition constant (K I 0.5 ) can now be termed as an inverse-related affinity constant (K′ D I-R ) for angiogenesis with a value of 3.2 pM which contrasts with the direct-related affinity constant (K′ D ) for osteoinduction at 7.3 nM.Since the constants of the two activities are separated by three and more orders of magnitude, identical receptors, shared, cross-reactive receptors and homologous desensitization (Popovic and Wilson, 2010) of receptors are improbable, strongly indicating an angiogenic receptor with constitutive activity (Berg and Clarke, 2018) (see saturation in Figure 4) and rhBMP-2 COL as a full inverse agonist in a typical dose response curve in Figure 4.
Control of local picomolar concentrations of rhBMP-2 COL in solute form (Table 4) are short-term and difficult, but via solidstate technology, e.g., as rhBMP-2 COL -PDLLA biohybrids (Figure 2; Table 3), are simple and long-term by sustained release (Table 2), comparable to a pH-Stat.In this study, not the pH but the growth factor concentration in the pico-or subpicomolar range is maintained as constant and guaranteed by high-affinity dissociation constants K′ D (= Pico-Stat) (Jennissen, 2023).

rhBMP-2 an angiogenesis inhibitor
As shown, the microvessel control values in the absence of rhBMP-2 (experiments of Tables 3, 4) are not zero but exhibit a significant spontaneous, endogenous pro-angiogenic control activity in the OEC/pOB cultures by VEGF-A (Dohle et al., 2021).This endogenous activity is inhibited by both species of rhBMP-2 (Dohle et al., 2021), for rhBMP-2 COL by concentrations above 10-20 pM (see Figures 3, 4).Thus it could be argued that the observed proangiogenic activity of rhBMP-2 COL (Figures 2,  3) is not of a stimulatory but only of a deinhibitory nature on dilution.However such a "deinhibition" neither accounts for a 9-fold net higher angiogenic biohybrid activity in vitro (Table 3) nor for a severalfold net higher vessel density in vivo (21 days, rats) in low versus high load comparison (Al-Maawi et al., 2023) (see Tables 1, 3).The above paradox angiogenic activity agrees with the evidence of rhBMP-2 COL being a full inverse agonist of a novel allosteric receptor complex together with an as yet unknown orthosteric agonist (see: Berg and Clarke, 2018;de Vries et al., 2021).

FIGURE 4
Dose-response curves of rhBMP-2COL for the induction of angiogenic (inverse agonist) and osteogenic activities.Cocultures of OEC/pOB cells (Dohle et al., 2021) and mono-cultures of MC3T3-E1 cells (Laub et al., 2007) were employed respectively with corresponding affinity constants K′ I D (see also legends to Table 4 and Figure 3).
Adsorbate load-dependent pro-and anti-angiogenic functions of the considered inverse agonist rh-BMP-2 COL on endothelial cells, has been determined by the novel method of solid-state biohybrids as nano-or pico-stats.
osteogenesis requiring angiogenesis, with the ingrowth of mesenchymal stem cells and osteoblasts.The global market size (MRI, 2023) of the recombinant form (rhBMP-2) [INFUSE ® Bone Graft (Food and Drug Administration, 2004)] reached USD 498.1 million in 2021

FIGURE 3
FIGURE 3Dose-response-curve of angioinduction by rhBMP-2COL after 7 days in OEC/pOB co-cultures.Inverse concentration dependence, half-maximal autoinhibition constant K I 0.5 , and a hypothetical K′D on a hypothetical dotted association line are shown (see Table4).
FIGURE 3Dose-response-curve of angioinduction by rhBMP-2COL after 7 days in OEC/pOB co-cultures.Inverse concentration dependence, half-maximal autoinhibition constant K I 0.5 , and a hypothetical K′D on a hypothetical dotted association line are shown (see Table4).

TABLE 3
Length and knot data of capillary-like structures induced by rhBMP-2COL adsorbate biohybrids after 14 days in co-culture*.

TABLE 4
Dose-response angioinduction in OEC/pOB co-cultures after 7 days by decreasing concentrations of rhBMP-2COL in solution*.